This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Czymmek, K. J. Articles by Howard, R. J. Search for Related Content PubMed Articles by Czymmek, K. J. Articles by Howard, R. J. Agricola Articles by Czymmek, K. J. Articles by Howard, R. J.

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

     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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Green fluorescent protein (GFP) is a 238 amino acid peptide isolated from the jellyfish Aequorea victoria that requires no exogenous substrates or co-factors for fluorescence (Chalfie et al 1994 ). Using standard molecular techniques, GFP can be utilized readily as a reporter for gene expression or as a fusion protein to monitor specific protein localization in living cells. The spectral properties of wild type GFP fluorescence include two excitation maxima, 395 and 475 nm, with a single emission maximum of 508 nm. Although successful expression of wild type GFP has been observed in a number of prokaryotic and eukaryotic organisms, many investigators have reported difficulty in detecting green fluorescence following transformation in filamentous fungi (Cormack 1998 , Fennandez-Abalos et al 1998 , Spellig et al 1996 , Suelmann et al 1998 ). Since GFP introduction, a variety of spectral and codon optimized mutants have been developed (Heim and Tsien 1996 ) together with the newer red fluorescent protein (RFP) isolated from the sea-anemone relative Discosoma sp. (Matz et al 1999 ). These fluorescent protein variants are well suited for use with common excitation wavelengths of laser scanning confocal and multiphoton microscopes.

The ability to monitor gene expression or visualize specific tagged proteins in vitro and in vivo has significant implications for fungal cell biology, and for the study of host-pathogen interactions in particular. Plants and fungi have chemically diverse cell walls that represent potent barriers to the introduction of antibody or other affinity probes. Optimal protocols for probe penetration into fungal cells (Bourett et al 1998 , Czymmek et al 1996 ) typically are not suitable for plants (Skalamera and Heath 1998 ). Over the last few decades, investigators have sought to overcome these inherent limitations in studies of host-plant fungal-pathogen interactions (Hardham and Mitchell 1998 ). Cytologists have relied on electron microscopy and/or light microscopy with sectioning, enzymatic digestion or harsh extraction methodologies in conjunction with general stains or other affinity probes to elucidate details of pathogenesis. Although many important insights have been gained through such studies, details of the infection process and quantitative three-dimensional (3-D) cytological data, particularly at later stages of infection, are difficult to follow and/or tedious or impossible to document.

In recent years, several investigators have succeeded in transforming economically important filamentous phytopathogens using GFP as a constitutively expressed reporter gene (Bowyer et al 2000 , Spellig et al 1996 , Stephenson et al 2000 , van West et al 1999 ). In addition, there have been numerous examples of GFP utilization in a variety of plants (Boevink et al 1998 , Marc et al 1998 , Nielsen et al 1999 , Reichel et al 1996 , Sheen et al 1995 , Shirasu et al 1999 ). The use of GFP not only circumvents many of the difficulties associated with the introduction of affinity probes into walled cells of plants and fungi, but also allows time resolved 3-D visualization of fungal infection and host responses in living tissues.

In this study, we examined interactions between Magnaporthe grisea and a susceptible cultivar of barley using cytoplasm-targeted, constitutively expressed fluorescent proteins. Specifically, we documented the cell-cell interaction, between host and pathogen during the infection process in 3-D over time—what is referred to as four-dimensional (4-D) imaging—using confocal or multiphoton microscopy. To our knowledge this is the first such report, other than our preliminary findings (Bourett et al 2000 ). Here we demonstrate how this new approach can provide novel information and a critical foundation for understanding the infection process. We believe that these methods will significantly aid studies of the molecular mechanisms of fungal pathogenicity and host resistance, and will be broadly applicable to other host-pathogen systems.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
\i\Fungal strains and transformation—Magnaporthe grisea \r\strains were maintained and propagated as previously reported (Valent et al 1991 ). Strains used in this study are described in Table I . Protoplast transformation was via the method of Sweigard et al (1995) . Transformants were selected using Bialophos (Cresent Chemical Co., Inc., Hauppauge, NY) and hygromycin B (Sigma, St. Louis, MO) resistance as selectable markers (DeZwaan et al 1999 ).

\i\Microscopy—\r\For initial screening of transformants and subsequent subcellular analysis of expression, we examined vegetative hyphae growing on a thin layer of defined complex medium (1% glucose, 0.17% yeast nitrogen base, 0.1% ammonium nitrate, 0.2% asparagine, pH 7.0) on the coverglass surface in a single-well coverglass chamber (Lab-Tek II chambered #1.5 coverglass, Nalge Nunc International, Naperville, IL). At least five transformants were selected for each fluorescent protein variant (except for EBFP transformants), based on fluorescence emission following excitation with the appropriate laser line. The brightest transformant from each set was used in subsequent trials (Table I ).

Appressoria were induced to form in vitro on the glass surface of a coverglass chamber by adding a drop of conidial suspension (5–10 x 10⁵ conidia/mL) containing 5 µg/mL 1,16-hexadecanediol and 0.1% DMSO in distilled water (DeZwaan et al 1999 ). Incipient appressoria were observed 4–6 h after inoculation.

To examine fungal structures in planta, intact leaves from 8–12 d barley plants cv BarSoy were point-inoculated with 10–20 µL of a conidial suspension (5–10 x 10⁴ conidia/mL) in distilled water of either strain MG134 (EGFP-expressing) or MG139 (EYFP-expressing), and incubated in a moist chamber until examination. Just prior to imaging, a 4 cm long piece of leaf was excised from the plant and attached carefully to the coverglass of a coverglass chamber using double-sided sticky tape, retaining a very thin layer of liquid between the coverglass and leaf surface.

Confocal images were acquired on inverted Zeiss LSM 410 or 510 confocal laser scanning microscopes (Carl Zeiss Inc., Thornwood, NY) using either a 40x (NA 1.2) or 63x (NA 1.2) water immersion Zeiss C-Apochromat objective lens. Confocal data were acquired using the following combination of laser lines and emission filters: for ECFP, the 458 nm laser line of a 10 mW argon laser (Omnichrome, Chino, CA) and 460–500 nm bandpass emission filter; for EGFP and EYFP, the 488 nm laser line of a 15 mW krypton:argon laser (Coherent, Inc., Santa Clara, CA) and a 500–550 bandpass; for RFP (DsRed) the 568 nm Coherent laser line, and a 590 nm longpass emission filter. Attempts to image EBFP were conducted using the 364 nm line from a UV laser (Coherent) and a 400 nm longpass emission filter. For continuous long-term imaging of living samples, laser powers typically were attenuated using an acusto-optic tunable filter to an average laser light power of 8–20 µW of laser light at the specimen. Fluorescence of EGFP- and EYFP-expressing infection structures, in combination with chloroplasts, were collected simultaneously, in two channels, using 488 nm excitation with 500–550 nm bandpass and 590 nm longpass filters, respectively.

Multiphoton data were acquired from EGFP expressing transformants using a 40x Plan-NeoFluar (NA 1.3) oil immersion objective on a Zeiss 510 NLO tuned to a 970 nm excitation wavelength on a Mira 900F femtosecond mode-locked titanium:sapphire laser (Coherent).

Images were captured as single optical sections (2-D), a z-series of optical sections (3-D), or a z-series over time (4-D). For renderings, 3-D and 4-D data sets were displayed as single red/green stereo anaglyphs or maximum intensity projections generated using Zeiss LSM software.

\i\Plant infection assays—\r\Whole-plant pathogenesis assays were conducted and results assessed according to methods previously described (DeZwaan et al 1999 ). Briefly, 1 wk barley plants were sprayed with a conidial suspension (7.5 x 10⁵ conidia/mL) from 8–14 d sporulating culture of each transformant. Pathogenicity was assessed 5 d following inoculation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Transformation of M. grisea with plasmids encoding GFP spectral variants resulted in high expression, with the exception of EBFP where no signal above background autofluorescence was detected. Constitutive expression of ECFP, EGFP and EYFP, under the control of the M. grisea ribosomal protein 27 promotor, resulted in extremely bright cytoplasmic fluorescence (Fig. 1 ). In all cases fluorescence was excluded from large organelles such as vacuoles and mitochondria (Fig. 1 ), but accumulated in interphase nuclei. Although fluorescence was also detected following transformation with Zm-GFP (Fig. 2 ), it was substantially weaker when signals were compared under identical growth and confocal imaging conditions (cf. Figs. 1, 2 ). To minimize the average laser power required for imaging, the brightest transformants for each fluorescent protein were selected. We found that under the imaging conditions described, laser power could be sufficiently attenuated to allow at least 7 h of continuous imaging of fluorescent protein-labeled fungal cells without any detectable photobleaching or laser induced cellular artifacts.

View larger version (49K): [in this window] [in a new window]    FIGS. 1–5. Imaging cytoplasm-targeted fluorescent proteins in living hyphae of Magnaporthe grisea. Comparison of fluorescence intensity of EGFP (FIG. 1 ) and Zm-GFP (FIG. 2 ) under the control of the same promotor. Single, median optical sections imaged under the identical conditions and microscope settings. FIGS. 3–5 . A series of single optical sections of a hypha expressing cytoplasmic RFP, imaged at three times during the mitotic cycle: interphase (FIG. 3 ), prophase (FIG. 4 ), and telophase (FIG. 5 ). During interphase the nucleus (N) showed RFP fluorescence, but the intensity was lower than cytoplasmic fluorescence and much lower than the bright fluorescence associated with condensed chromatin during mitosis (FIGS. 4, 5 , arrows). Bar = 5 µm

View larger version (23K): [in this window] [in a new window]    FIG. 6. Simultaneous visualization of EGFP- and RFP-expressing incipient appressoria of M. grisea induced on a glass surface in the presence of 1,16-hexadecanediol. Set of 30 optical sections rendered as a maximum intensity projection. Note the accumulation of RFP in vacuoles of the two conidia (arrows). Bar = 10 µm

Since plant pathogenic interactions are inherently 4-D, we used a 4-D approach for data acquisition. A stack of optical sections (i.e., z-series) was taken repeatedly using the laser scanning confocal microscope, in the same location over time. Depending on the conditions and time interval between data sets, 4-D data reflected volumetric and/or subcellular changes on the order of hours (Figs. 7–10 ), minutes (Figs. 12–24 ), or seconds (not shown). An example of a 4-D data set is illustrated in Figs 7–10 , where five time points during fungal pathogenesis of a barley leaf with an EGFP-expressing M. grisea transformant are displayed as a series of red/green stereo anaglyphs. Following appressorial penetration of the upper epidermal leaf surface, the behavior and fate of intracellular infection hyphae were observed readily within epidermal cells. Of particular note in this series were contrasting successful and failed infections visible at different encounter sites (Figs. 7–10 ): two thriving colonies appear in the same vicinity as another where infection hyphae died after ca 70 h as indicated by cessation of growth and concomitant loss of EGFP fluorescence (cf. Figs. 9, 10 , arrows). This loss of fluorescence, varying fluorescence intensity, and variable growth rates among the infection hyphae can be viewed as a movie at the following URL: http://www.udel.edu/bio/information/facilities/microscopy/avi/. Figures 7–11 are stereo anaglyphs. The stereo effect can only be seen through red/green stereo glasses. A pair of stereo glasses may be obtained from the authors at no charge.

View larger version (42K): [in this window] [in a new window]    FIGS. 7–11. To be viewed through red-green stereo glasses, these anaglyphs demonstrate in planta imaging of the interaction between an EGFP-expressing M. grisea transformant and susceptible barley leaf. Images represent part of a 3-D time series captured over a 31 h period at 40 (FIG. 7 ), 51 (FIG. 8 ), 61 (FIG. 9 ) and 71 h post-inoculation (FIG. 10 ). Infection hyphae, produced from appressoria (arrows) formed on the leaf surface, grew intracellularly within epidermal cells. Infection hyphae from one encounter site were apparently unsuccessful as evidenced by cessation of growth and a significant reduction in EGFP fluorescence between 61 and 71 h post-inoculation (arrows). Bar = 20 µm FIG. 11 . Subsequent fixation of the infected leaf with glutaraldehyde increased autofluorescence, and thereby facilitated visualization of plant cells. Bar =10 µm. [AVI movie of these data can be downloaded at http://www.udel.edu/bio/information/facilities/microscopy/avi]. NOTE: stereo glasses are to be worn with green filter over right eye.

View larger version (74K): [in this window] [in a new window]    FIGS. 12–26. Confocal (FIGS. 12–24, 26 ) and multiphoton (FIG. 25 ) imaging of susceptible barley leaves infected by a M. grisea transformant expressing either EYFP (FIGS. 12–24 ) or EGFP (FIGS. 25, 26 ). [Note: AVI movies of the in vivo imaging can be downloaded at http://www.udel.edu/bio/information/facilities/microscopy/avi/]. FIGS. 12–19 . Dual channel in vivo imaging reveals fungal (green) penetration of a mesophyll cell delimited by peripheral chloroplasts (red). A single chloroplast (C) is displaced (cf. FIGS. 13, 14 ) as the fungus enters the host cell (arrows). Bar = 5 µm. FIGS. 20–24 . At a different encounter site, fungal penetration (arrows) resulted in collapse of the host mesophyll cell (cf. FIGS. 21, 23 ). An intercellular infection hypha (IH) was observed to grow past the infected mesophyll cell at a much faster rate. These dual channel image series represent line-averaged, maximum intensity projections recorded continuously, with a 6.25 min interval per image. Bar = 5 µm. FIG. 25 . Multiphoton imaging following glutaraldehyde fixation illustrates well-resolved fungal infection hyphae within host epidermal cells. Very bright, intermittent fluorescence emission was associated with melanized appressoria (arrows), and the region of the penetration peg (arrowheads). Maximum intensity projection of 15 optical sections at a z-interval of 0.6 µm. Bar = 10 µm. FIG. 26 . Autofluorescent bodies (arrows) were observed in mesophyll cells, only in infected tissue, after glutaraldehyde fixation and dual channel confocal imaging. Single optical section. Bar = 20 µm

Because red chloroplast autofluorescence could be separated spectrally from EGFP emission, dual channel imaging was used to examine later stages of infection when visualization of peripheral chloroplasts facilitated the recognition and demarcation of mesophyll cells in living tissue (Figs. 12–24 ). The process of an infection hypha exiting one cell and entering another, or exiting into extracellular spaces, varied markedly among encounter sites for the compatible host-pathogen interaction studied here. Some extracellular infection structures of M. grisea penetrated mesophyll cells relatively slowly via an apparently rigid penetration peg-like structure of narrower diameter than infection hyphae, without host cell collapse. Entry of the pathogen could be noted, for example, by the displacement of a chloroplast (Figs. 12–19 , arrows). In other examples of host cell penetration from an extracellular space, more rapid penetration and extension of an infection hypha was soon followed by collapse of the host cell (Figs. 20–24 )—presumably due to loss of plant cell turgor. Examples of such 4-D sequences can be viewed as a movie at the same URL given above: http://www.udel.edu/bio/information/facilities/microscopy/avi/. Typically, very rapid infection hypha growth and penetration were prerequisites for loss of host cell turgor, although heavily populated individual plant cells also exhibited this phenomenon.

Multiphoton microscopy was also used to visualize host-pathogen encounter sites using EGFP expressing transformants in living and fixed (Fig. 25 ) tissue. This approach permitted improved imaging deeper within plant tissue. Near-infrared excitation of melanized appressoria resulted in a very unusual emission pattern of very high intensity and clearly non-uniform in median optical sections of appressoria (Fig. 25 ). Similar emission was observed also in the region of the penetration peg (Fig. 25 , arrows).

Small spherical fluorescent bodies up to 5 µm in diameter appeared within mesophyll cells during later stages of the infection process (Fig. 26 ), especially in cells along the periphery of a lesion. These bodies were not observed in uninfected tissue. As determined by 3-D analysis, these fluorescent bodies were typically located in the peripheral cytoplasm of mesophyll cells adjacent to abutting epidermal cells. In vivo cell imaging indicated that these fluorescent bodies often exhibited saltatory movement within mesophyll cells prior to the onset of close contact with fungal infection hyphae. Following this interaction, movement often appeared to cease and the fluorescent bodies became juxtaposed with the extracellular fungal structures. There was a strong yellow/orange emission with 488nm excitation, and weak red emission using 568 nm excitation. The fluorescence intensity of these bodies increased following fixation with glutaraldehyde (Fig. 26 ), and the emission spectrum appeared to vary among individual bodies. Within the same cell, there was often one large fluorescent body with several smaller satellite bodies in proximity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
There has been a rapid expansion in the use of GFP as a molecular marker for fungal related applications during the last few years. The broad range of fluorescent protein applications in filamentous fungi is a strong indication of the versatility of the approach. GFP has been used to study cytoskeletal elements (Lehmler et al 1997 , Xiang et al 2000 , Yamashita et al 2000 ), pathogenicity-related genes (DeZwaan et al 1999 , Dumas et al 1999 , Spellig et al 1996 , Zeilinger et al 1999 ), genes involved in general biochemistry, growth and differentiation (Bae and Knudsen 2000 , Berteaux-Lecellier 1998 , Valdez-Taubas et al 2000 , Vautard-Mey et al 1999 ), and subcellular targeting (Fernandez-Abalos et al 1998 , Gordon et al 2000 , Siedenberg et al 1999 , Sievers et al 1999 , Suelmann et al 1998 , Suelmann and Fischer 2000 ). The utility of GFP as a vital reporter has been realized in the vast majority of these studies, as unfixed samples were viewed both in vitro and in vivo.

We believe that the use of fluorescent proteins and confocal imaging to obtain 4-D data from living tissue complements other techniques and represents a powerful approach for elucidating details of plant-fungal interactions at a microscopic level. Significant advances in the latest generation of confocal microscopes—including increased sensitivity, better detectors, shorter light paths, better optical coatings and filters, longer working distance water-immersion objective lenses, use of an acusto optic filter for precise laser light control, four-dimensional acquisition and analysis software—have enabled imaging that was difficult or impossible just a few years ago. Confocal and multiphoton microscopy are ideally suited for fluorescent imaging of plant-fungal interactions due to non-invasive optical sectioning properties that dramatically reduce out-of-focus information from the final image (Czymmek et al 1994 , Duncan and Howard 2000 , Howard 2001 , König 2000 ).

Despite these advantages, thus far there have been only a few reports of in vivo confocal microscopy of plant-fungal interactions. Vierheilig et al (1999) imaged arbuscular mycorrhizal structures in roots using their inherent autofluorescence. Bowyer et al (2000) used a GFP fusion to analyze changes in carbon metabolism during infection of wheat by Tapesia yallundae. Stephenson et al (2000) and DeZwaan et al (1999) used GFP-promoter fusion proteins to monitor expression of pathogenicity genes during host infection.

Over the last few decades several microscopy methods have been applied to examine the interactions between host cells and the blast pathogen, Magnaporthe grisea. Light microscope protocols for imaging the fungus in fixed material include sectioning methods (Kawamura 1940 ), staining with colorimetric (Peng and Shishiyama 1988 ) and fluorescent dyes (DeZwaan et al 1999 , Koga et al 1986 ), and chloral hydrate clearing of samples for analysis using differential interference contrast optics (Heath et al 1990 ). The latter is a very useful technique—both fungus and plant cells can be observed easily, and samples can be sectioned optically—but drawbacks include the temporally static nature of the image, and the inability to generate meaningful 3-D renderings. Examination of living infected plant tissue during compatible and incompatible interactions has been accomplished using epidermal strips (Koga 1994 ). This approach was used to improve imaging clarity for both conventional transmitted and fluorescence light optics. Koga's study was the first to provide temporal information regarding individual M. grisea penetration events, however fungal structures within intact plant organs, including leaves, could not be imaged.

Although we found many new details concerning pathogenesis during a compatible interaction, it must be noted that there were a few technical difficulties that needed to be overcome for successful 4-D imaging. For example, sample drift was problematic over long imaging periods, so a method was devised to attach and view a number of plant tissue types in a humid chamber that minimized sample perturbation and allowed free gas exchange. These conditions tended to minimize sample drift during data acquisition over many hours, however imaging parameters such as focal plane were modified as necessary during each experiment.

Reasonable caution was exercised when imaging samples for long periods of time under continuous laser illumination for a number of reasons. Excessive laser light may cause artifacts with either plant or fungal cell physiology, due for example to local heating or blue-light mediated events. Significant photobleaching of signal will prevent longer or more frequent data collection, and phototoxic effects (if any) will be greater at higher laser powers. We did not notice any deleterious effects during prolonged, continuous imaging of fluorescent protein-expressing transformants. We believe this was facilitated by the use of very low but sufficient laser intensity, sufficient because of a combination of very strong fluorescence signal from transformants and high sensitivity of our laser scanning confocal microscope. Laser light was attenuated using an acusto optic tunable filter to a power resulting in 8–20 µW of light at the specimen. This range was more than an order of magnitude lower than that found to cause transient disturbances in plasma membrane of Arabidopsis roots (Tirlapur and König 1999 ). We found that these laser powers were low enough for continuous imaging of GFP-labeled fungal cells for at least 7 h without detectable adverse effects on the specimen. Since photobleaching was negligible under these conditions, the need for anti-fade compounds was eliminated.

The autofluorescent plant bodies that were observed only in infected tissues, could be composed of anthocyanins and/or phenolic compounds, were excited at 488 nm and were distinguished clearly from the EGFP and chloroplast emission signals excited by the same laser line. These bodies may be an important indicator of host response during the disease process. The timing of their appearance (i.e., visible autofluorescence), location within mesophyll cells, and non-random alignment along upper epidermal cells, suggest some relationship with a general host defense response. Others have described the induction of fluorescent plant compounds in response to pathogen attack, but published images reveal a massive cell-wide accumulation of fluorescent compounds that are generally thought to be associated with the hypersensitive response (see review by Heath 2000 ). Although they did not use fluorescence, Snyder and Nicholson (1990) describe an accumulation of pigmented phytoalexin bodies in epidermal cells of sorghum, in response to fungal attack, with similar morphology and behavior as the autofluorescent bodies reported here.

In addition to these autofluorescent bodies, we observed many details of in planta infection at specific encounter sites with M. grisea transformants expressing cytoplasmic fluorescent proteins. These included 1) collapse of plant cells under certain conditions, 2) differential growth rates of inter- and intracellular fungal infection hyphae, 3) heterogeneity in growth rate and morphology of infection hyphae during penetration, 4) sudden loss of fluorescence in hyphae, presumably indicative of a successful host resistance response, and 5) gradual decrease in the fluorescence intensity of older infection hyphae over time, suggestive of a decline in cellular metabolism.

Multiphoton microscopy was a viable alternative to confocal for imaging fluorescent protein-expressing fungi during pathogenesis. As expected, the near infrared excitation used in multiphoton imaging improved observation deeper within plant tissue. Barley leaves were highly prone to scattering visible laser light as it passed through plant cell walls and at interfaces with intercellular air spaces.

We have demonstrated the feasibility of a multi-color approach for monitoring aspects of plant pathogenesis with currently available fluorescent proteins. The fact that all of this information was extracted from living tissue has important implications for studies of plant-fungal interactions as we discover new genes that mediate the infection process. The present study has provided much new information regarding a single compatible interaction. We expect that studies employing fluorescent proteins in the form of protein fusions will be even more useful in a whole system approach, especially where transformation of both host and pathogen is possible.

	FOOTNOTES

 
¹ Corresponding author, Email: Richard.J.Howard{at}usa.dupont.com

Accepted for publication August 26, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Bae Y, Knudsen GR., 2000 Cotransformation of Trichoderma harzianum with ß-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils Environ Microbiol 66:810-815

Berteaux-Lecellier V, Zickler D, Debuchy R, Panvier-Adoutte A, Thompson-Coffe C, Picard M., 1998 A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina EMBO J 17:1248-1258[Medline]

Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C., 1998 Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network Plant J 15:441-447[Medline]

Bourett TM, Czymmek KJ, Dezwaan TM, Sweigard JA, Howard RJ., 2000 Localization and visualization of specific gene products in fungal plant pathogens and their hosts Microsc Microanal 6:680-681

Bourett TM, Czymmek KJ, Howard RJ., 1998 An improved method for affinity probe localization in whole cells of filamentous fungi Fungal Genet Biol 24:3-13[Medline]

Bowyer P, Mueller E, Lucas J., 2000 Use of an isocitrate lyase promoter-GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of wheat Mol Plant Pathol 1:253-262

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC., 1994 Green fluorescent protein as a marker for gene expression Science 263:802-805[Abstract/Free Full Text]

Cormack B., 1998 Green fluorescent protein as a reporter of transcription and protein localization in fungi Curr Opin Microbiol 1:406-410[Medline]

Czymmek KJ, Bourett TM, Howard RJ., 1996 Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution and methacrylate de-embedment J Microsc 181:153-161

Czymmek KJ, Whallon JH, Klomparens KL., 1994 Confocal microscopy in mycological research Exp Mycol 18:275-293.

DeZwaan TM, Carroll AM, Valent B, Sweigard JA., 1999 Magnaporthe grisea Pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues Plant Cell 11:2013-2030[Abstract/Free Full Text]

Dumas B, Centis S, Sarrazin N, Esquerre-Tugaye M-T., 1999 Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection Appl Environ Microbiol 65:1769-1771[Abstract/Free Full Text]

Duncan KE, Howard RJ., 2000 Cytological analysis of wheat infection by the leaf blotch pathogen Mycosphaerella graminicola Mycol Res 104:1074-1082

Fernandez-Abalos JM, Fox H, Pitt C, Wells B, Doonan JH., 1998 Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans Mol Microbiol 27:121-130[Medline]

Gordon CL, Archer DB, Jeenes DJ, Doonan JH, Wells B, Trinci APJ, Robson GD., 2000 A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger J Microbiol Methods 42:39-48[Medline]

Hardham AR, Mitchell HJ., 1998 Use of molecular cytology to study the structure and biology of phytopathogenic and mycorrhizal fungi Fungal Genet Biol 24:252-284

Heath MC., 2000 Hypersensitive response-related death Plant Mol Biol 44:321-334[Medline]

Heath MC, Valent B, Howard RJ, Chumley FG., 1990 Correlations between cytologically detected plant-fungal interactions and pathogenicity of Magnaporthe grisea toward weeping lovegrass Phytopathology 80:1382-1386.

Heim R, Tsien RY., 1996 Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer Curr Biol 6:178-182[Medline]

Howard RJ., 2001 Cytology of fungal pathogens and plant-host interactions Curr Opin Microbiol 4:365-373[Medline]

Kawamura E., 1940 Reaction of certain species of the genus Oryza to the infection of Pyricularia oryzae Bull Sci Fac Terkult Kyusyu Imp Univ 9:157-166

Koga H., 1994 Hypersensitive death, autofluorescence, and ultrastructural changes in cells of leaf sheaths of susceptible and resistant near-isogenic lines of rice (Pi-z^t) in relation to penetration and growth of Pyricularia oryzae Can J Bot 72:1463-1477

Koga H, Horino O, Kobayashi T., 1986 Ultrastructural studies on the resistance mechanisms of rice plants infected with Pyricularia oryzae Cav Bull Hok Nation Agr Exp Sta 28:1-56

König K., 2000 Multiphoton microscopy in life sciences J Microsc 200:83-104[Medline]

Lehmler C, Steinberg G, Snetselaar KM, Schliwa M, Kahmann R, Bolker M., 1997 Identification of a motor protein required for filamentous growth in Ustilago maydis EMBO J 16:3464-3473[Medline]

Marc J, Granger CL, Brincat J, Fisher D, Kao T, McCubbin AG, Cyr RJ., 1998 A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells Plant Cell 10:1927-1939[Abstract/Free Full Text]

Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA., 1999 Fluorescent proteins from nonbioluminescent Anthozoa species Nature Biotechnol 17:969-973[Medline]

Nielsen K, Olsen O, Oliver R., 1999 A transient expression system to assay putative antifungal genes on powdery mildew infected barley leaves Physiol Mol Plant Pathol 54:1-12

Oldenburg KR, Vo KT, Michaelis S, Paddon C., 1997 Recombination-mediated PCR-directed plasmid construction in vivo in yeast Nucleic Acids Res 25:451-452[Abstract/Free Full Text]

Peng YL, Shishiyama J., 1988 Temporal sequence of cytological events in rice leaves infected with Pyricularia oryzae Can J Bot 66:730-735

Reichel C, Mathur J, Eckes P, Langenkemper K, Koncz C, Schell J, Reiss B, Maas C., 1996 Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells Proc Natl Acad Sci USA 93:5888-5893.[Abstract/Free Full Text]

Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW., 1995 Green-fluorescent protein as a new vital marker in plant cells Plant J 8:777-784[Medline]

Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P., 1999 Cell-autonomous complementation of mlo resistance using a biolistic transient expression system Plant J 17:293-299

Siedenberg D, Mestric S, Ganzlin M, Schmidt M, Punt PJ, van den Hondel CAMJJ, Rinas U., 1999 GlaA promoter controlled production of a mutant green fluorescent protein (S65T) by recombinant Aspergillus niger during growth on defined medium in batch and fed-batch cultures Biotechnol Prog 15:43-50[Medline]

Sievers N, Bertsch E, Fischer R., 1999 Isolation of nuclear migration mutants of Aspergillus nidulans using GFP expressing strains Mycol Res 103:961-966

Skalamera D, Heath MC., 1998 Changes in the cytoskeleton accompanying infection-induced nuclear movements and the hypersensitive response in plant cells invaded by rust fungi Plant J 16:191-200

Snyder BA, Nicholson RL., 1990 Synthesis of phytoalexins in sorghum as a site-specific response to fungal ingress Science 248:1637-1639[Abstract/Free Full Text]

Spellig T, Bottin A, Kahmann R., 1996 Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis Mol Gen Genet 252:503-509[Medline]

Stephenson SA, Hatfield J, Rusu AG, Maclean DJ, Manners JM., 2000 CgDN3: an essential pathogenicity gene of Colletotrichum gloesporiodes necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis Mol Plant Microbe Interact 13:929-941[Medline]

Suelmann R, Sievers N, Galetzka D, Robertson L, Timberlake WE, Fischer R., 1998 Increased nuclear traffic chaos in hyphae of Aspergillus nidulans: molecular characterization of aspB and in vivo observation of nuclear behaviour Mol Microbiol 30:831-842[Medline]

———, Fischer R., 2000 Mitochondrial movement and morphology depend on an intact actin cytoskeleton in Aspergillus nidulans Cell Motil Cytoskeleton 45:42-50[Medline]

Sweigard J, Carroll A, Kang S, Farrall L, Chumley F, Valent B., 1995 Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus Plant Cell 7:1221-1233[Abstract]

Tirlapur UK, König K., 1999 Near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3-D imaging of localized dye-coupling in the Arabidopsis root meristem Plant J 20:363-370[Medline]

Valdez-Taubas J, Diallinas G, Scazzocchio C, Rosa AL., 2000 Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans Fungal Genet Biol 30:105-113[Medline]

Valent B, Farrall L, Chumley F., 1991 Magnaporthe grisea genes for pathogenicity and virulence identified through a series of backcrosses Genetics 127:87-101[Abstract]

van West P, Reid B, Campbell TA, Sandrock RW, Fry WE, Kamoun S, Gow NAR., 1999 Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora FEMS Microbiol Lett 178:71-80[Medline]

Vautard-Mey G, Cotton P, Fevre M., 1999 Expression and compartmentation of the glucose repressor CRE1 from the phytopathogenic fungus Sclerotinia sclerotiorum Eur J Biochem 266:252-259[Medline]

Vierheilig H, Bockenhoff A, Knoblauch M, Juge C, Van Bel AJE, Grundler F, Piche Y, Wyss U., 1999 In vivo observations of the arbuscular mycorrhizal fungus Glomus mosseae in roots by confocal laser scanning microscopy Mycol Res 103:311-314

Xiang X, Han G, Winkelmann DA, Zuo W, Morris NR., 2000 Dynamics of cytoplasmic dynein in living cells and the effect of mutation in the dynactin complex actin-related protein Arp1 Curr Biol 10:603-606[Medline]

Yamashita RA, Osherov N, May GS., 2000 Localization of wildtype and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins Cell Motil Cytoskeleton 45:163-172[Medline]

Zeilinger S, Galhaup C, Payer K, Woo SL, Mach RL, Fekete C, Lorito M, Kubicek CP., 1999 Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host Fungal Genet Biol 26:131-140[Medline]

This article has been cited by other articles:

				P. Kankanala, K. Czymmek, and B. Valent Roles for Rice Membrane Dynamics and Plasmodesmata during Biotrophic Invasion by the Blast Fungus PLANT CELL, February 1, 2007; 19(2): 706 - 724. [Abstract] [Full Text] [PDF]

				R. M. Andrie, J. P. Martinez, and L. M. Ciuffetti Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes. Mycologia, September 1, 2005; 97(5): 1152 - 1161. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Czymmek, K. J. Articles by Howard, R. J. Search for Related Content PubMed Articles by Czymmek, K. J. Articles by Howard, R. J. Agricola Articles by Czymmek, K. J. Articles by Howard, R. J.