Plant Cell, Vol. 10, 585-598, April 1998, Copyright © 1998, American Society of Plant Physiologists
Role of Calcium in Signal Transduction during the Hypersensitive Response Caused by Basidiospore-Derived Infection of the Cowpea Rust Fungus
Haixin Xua and
Michèle C. Heatha
a Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada
Correspondence to:
Michèle C. Heath, heath{at}botany.utoronto.ca (E-mail), 416-978-5878 (fax).
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ABSTRACT |
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The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea–cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.
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INTRODUCTION |
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During their lifetime, plants are subjected to thousands of microbial attacks. Active plant defenses against these microbes include the formation of mechanical barriers and increases in the levels of potentially antimicrobial molecules (Lamb et al. 1989 
; Dixon et al. 1994 ) and are usually accompanied by the rapid death of one or more plant cells. This rapid, localized cell death associated with disease resistance is known as the hypersensitive response (HR). Increasing evidence indicates that the HR is a form of programmed cell death (Greenberg et al. 1994 ; Heath 1998 ) that has some features in common with mammalian apoptosis (Ryerson and Heath 1996 ; Mittler et al. 1997 ).
The initial requirement of any defense response is the perception of the pathogen by the plant. This recognition presumably leads to a signal transduction cascade in the plant cell (Ebel and Cosio 1994 ), but signal transduction pathways in plants have yet to be clearly elucidated. In plants, as in animals, many stimuli are mediated by elevation of cytosolic free calcium ([Ca2+]i) (Bush 1995 ). The involvement of calcium in responses of cell cultures or protoplasts to microbial products (elicitors) has been demonstrated (Mahady and Beecher 1994 ; Messiaen and Van Cutsem 1994 ; Suzuki et al. 1995 ; Tavernier et al. 1995 ; Ishihara et al. 1996 ; Levine et al. 1996 ), and patch–clamp experiments have indicated that elicitors may affect the activity of plasma membrane calcium channels (Gelli et al. 1997 ; Zimmermann et al. 1997 ). Nevertheless, although the responses of cell cultures or protoplasts to microbial elicitors represent convenient experimental systems for analysis of plant defense mechanisms, they do not provide information on the spatial orchestration of defenses that occurs in intact plants in relation to localized damage and pathogen ingress (Lamb et al. 1989 ).
By using calcium reporting dyes, the measurement of [Ca2+]i in intact tissues has been achieved with coleoptiles (Gehring et al. 1990 ) and root hairs (Ehrhardt et al. 1996 ) but has never been performed with cells during penetration by a pathogenic fungus. Therefore, we examined cytosolic calcium levels during the HR of epidermal cells in cowpea plants inoculated with basidiospores of the cowpea rust fungus (Uromyces vignae), an obligate biotrophic phytopathogen and the causal agent of cowpea rust disease. Cell invasion by race 1 of the cowpea rust fungus provokes an HR in resistant cowpea cultivar Dixie Cream but does not cause any detectable resistance response, or cell death, in a susceptible cultivar, California Blackeye (Heath 1989 ; Heath et al. 1997 ). By using confocal laser scanning microscopy in conjunction with a highly fluorescent calcium reporter dye, Calcium Green-1–dextran (CG-1) linked with Texas Red, we show here that an elevation of [Ca2+]i was detectable in epidermal cells of resistant plants before completed penetration of the plant epidermal wall and before other detectable cytoplasmic manifestations of the HR. The elevation of [Ca2+]i was prevented by calcium channel inhibitors, which also delay the HR. The data suggest that extracellular and possibly intracellular calcium stores may be involved in elevating [Ca2+]i and that calcium plays an important role in signal transduction leading to the HR.
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RESULTS |
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Fungal Infection and Plant Responses
When a basidiospore of U. vignae lands on the surface of a cowpea leaf, it typically germinates and forms a slightly swollen appressorium at the tip of a short germ tube. This appressorium forms a penetration peg, which can be seen under the light microscope as a tiny circle in the middle of the appressorium. As the peg grows through the plant wall, the plant nucleus migrates to the penetration site but leaves at about the time the fungus touches the plant plasma membrane (Heath et al. 1997 ). The fungus then forms a spherical intraepidermal vesicle within the epidermal cell that is separated from the host cytoplasm by an extension of the plant plasma membrane.
In susceptible cultivars, the fungus establishes tip growth to form a primary hypha, the plant nucleus becomes closely associated with the fungus, and the latter eventually grows into neighboring epidermal cells and into the leaf mesophyll below (Heath 1989 ; Xu and Mendgen 1991 ). This growth does not cause the death of any host cells. However, in the resistant cultivar, the plant nucleus commonly does not return to the fungus when the fungus establishes tip growth, and fungal growth is curtailed by the hypersensitive death of the first invaded cell. Various stages in hypersensitive cell death can be recognized cytologically. Early stages include a change in nuclear appearance, the cessation of cytoplasmic streaming, and the subsequent appearance of jiggling particles in the vacuole (Chen and Heath 1991 ; Heath et al. 1997 ).
Ratio Images of [Ca2+]i in Water-Incubated Tissue
To visualize changes in [Ca2+]i, we loaded the calcium-sensitive dye CG-1 and the calcium-insensitive dye Texas Red linked to the same dextran (10 kD) into individual epidermal cells by microinjection. Both dyes can be excited by visible light and are therefore less cytotoxic than indo-1 or fura-3, which require excitation by UV wavelengths. Dual fluorescent images were collected at the same time. One of the images was for CG-1 and indicated [Ca2+]i; the other was for Texas Red and indicated the dye concentration. The calculated ratio values indicate relative [Ca2+]i irrespective of the dye concentrations injected into the cells. This technique eliminates the need to inject a constant amount of dye into the cell, which is necessary when using nonratiometric Ca2+ indicators, such as CG-1 alone, that do not undergo a significant shift in emission wavelength when bound to calcium (Diliberto et al. 1994 ).
Conjugation of both dyes to the macromolecular dextran usually prevents sequestration by organelles. The dye concentrations loaded by pressure microinjection were estimated to be 10 to 20 µM in this study. These intracellular dye concentrations would not be expected to contribute significantly to the Ca2+-buffering capacity of the cytoplasm (Gilroy and Jones 1992 ).
To avoid artifacts for ratio analysis of [Ca2+]i, it is essential that the cell be injected successfully. A successfully injected cell was judged to be one in which cytoplasmic streaming occurred throughout the observation period and in which the dyes were excluded from the cell vacuole. Poor injection or loading stress led to vacuolar accumulation of the dyes within a few minutes, and these cells were not studied further. The injection needle was removed immediately after dye injection, probably accounting for the fact that microinjection did not induce callose papilla formation (data not shown), which is a typical and ubiquitous response to wounding (Aist 1976 ). Figure 1A illustrates the dual fluorescent images of confocal laser scanning microscopy of a single uninfected epidermal cell that was successfully injected. This cell is characterized by a non-uniform distribution of dyes that parallel the distribution of cytoplasm. No autofluorescence of epidermal cells was seen.
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Figure 1.
Dual Fluorescent Images of Confocal Laser Scanning Microscopy.
(A) Dual fluorescent images of an epidermal cell of a resistant plant injected and loaded with 10-kD dextran linked with CG-1 and Texas Red. At left is the image reported by CG-1 for [Ca2+]i; at right is the image reported by Texas Red for the dye concentration. The cytoplasmic strands and nuclear area clearly seen in this cell indicate that microinjection did not impair the viability of the cell. The neighboring cells of the dye-loaded cell did not show any autofluorescence.
(B) A set of dual images similar to that in (A). The boxed area shown at left was used to obtain the average number of photons per pixel for calculation of the numerical ratio values of fluorescence intensity.
Bars = 25 µm.
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Numerical ratio values were obtained by dividing the average number of photons per pixel in a boxed area covering the whole cell in the CG-1 image (as shown in Figure 1B) by the comparable value for the Texas Red image. Five to six values were taken from each cell over a 20- to 30-min period and averaged to compensate for any periodic fluctuations in calcium levels. Because fungal development is not synchronous, the stage of infection of each cell was identified by a combination of cytological features. During the early stages of invasion when the fungal penetration peg was still within the epidermal wall, relative fungal development was estimated by determining whether the plant nucleus was moving to, associated with, or moving away from the penetration site. The size of the fungal intraepidermal vesicle and whether the plant nucleus had migrated back to the fungus indicated the relative chronology once the fungus had penetrated the cell.
Table 1 provides the averaged values for individual cells of both the resistant and susceptible cultivars at different early stages of fungal invasion. Values for the susceptible cultivar are similar to those of uninfected cells when the fungus was growing through the plant wall; however, a higher value was recorded in one cell containing a small intraepidermal vesicle, and cells with larger intraepidermal vesicles had values just below that of uninfected cells. Means of values from cells with the fungus in the plant wall or from cells containing intraepidermal vesicles did not differ statistically (Student's t test; P > 0.05) from the mean from uninfected cells. Uninfected cells of the resistant cultivar had ratio values statistically identical to those of uninfected susceptible cells. However, values were strikingly higher in resistant Dixie Cream cells in which the fungal penetration peg was growing through the plant wall before entering the cell lumen. The means of these values were significantly different (Student's t test; P < 0.05) from that of uninfected resistant cells and from infected susceptible cells at the same stage of infection. Ratio values were still high in resistant cells with a small intraepidermal vesicle but diminished in cells with a larger intraepidermal vesicle, so the mean of the values from intraepidermal vesicle-containing resistant cells was not statistically different from that of uninfected resistant cells or intraepidermal vesicle-containing susceptible cells. From the in vitro calibration curve shown in Figure 2, it appears that the [Ca2+]i levels in the resistant cells rose from ~100 to ~600 nM; however, these values must be considered approximate because the dissociation constant of calcium indicators is dependent on factors such as pH and ionic strength, which are unknown in the living cell.
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Figure 2.
In Vitro Calibration Curve for Ratio Values versus Free Calcium Concentrations.
Fluorescence ratio values were obtained from calcium buffer solutions containing 10 µM CG-1 and Texas Red observed on a microscope slide. Values are the means ±SD of three or four measurements.
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Table 1.
Fluorescence Ratio Valuesa (CG-1/ Texas Red) for Dixie Cream and California Blackeye Epidermal Cells at Different Stages of Fungal Invasion
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Visual images of representative stages of fungal infection in resistant and susceptible cells, derived by pixel-by-pixel ratio analysis, are shown in Figure 3A to F. A representative time course of images illustrating the rise in [Ca2+]i in a single resistant cell as the fungus grows through the cell wall is shown in Figure 4. Figure 4 illustrates that it took ~9 min for the [Ca2+]i to rise from the base level to the maximum. Figure 4 also shows that although increases in [Ca2+]i in root hairs have been reported to start around the nuclear area (Ehrhardt et al. 1996 ), in this cell, as in the others examined, the increase in [Ca2+]i was not initiated in the nuclear region.
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Figure 3.
Visual Images of Pixel-by-Pixel Ratio Analysis Showing Typical Distribution of [Ca2+]i in Epidermal Cells.
(A) Uninfected resistant plant cell.
(B) Infected resistant plant cell with fungus growing through the plant wall.
(C) Infected resistant plant cell containing a large fungal intraepidermal vesicle.
(D) Uninfected susceptible plant cell.
(E) Infected susceptible plant cell with fungus in wall.
(F) Infected, susceptible plant cell containing a large intraepidermal vesicle.
[Ca2+]i has been arbitrarily coded according to the scale, where white (2.00) represents the maximum and black (0.00) the minimum [Ca2+]i; these numbers cannot be directly related to the numerical ratios shown in Table 1 Table 2 Table 3 and Figure 2, Figure 5, and Figure 6. Bars = 25 µm.
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Figure 4.
Spatial and Temporal Changes of [Ca2+]i in a Resistant Epidermal Cell.
Pixel-by-pixel ratio images show a representative time course from a single cell in which the fungus was growing through the plant wall. During the observation period, the plant nucleus (arrow) did not move from beneath the penetration site. The increase in [Ca2+]i was not initiated from the nuclear region. Numbers indicate the time in minutes after the first collected picture (5 to 10 min after injection of the dyes). The intensity scale for the relative [Ca2+]i is as described in Figure 1. Bar = 25 µm for all images.
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In all of the resistant cells examined, the plant cytoplasm was still streaming during observation, and in fungal-infected cells, there was no detectable sign of the onset of the HR at the rather low level of resolution provided by the confocal laser scanning microscope. Attempts to inject cells at an early stage of the death process when particles were seen jiggling in the plant vacuole (Heath et al. 1997 ) were not successful because the plant cytoplasm did not seem to be able to withstand the injection process.
Effect of Kinetin on the Elevation of [Ca2+]i
Detachment of plant leaves, which is necessary for cell injection and observation, can delay the hypersensitive death of resistant, invaded cells by several hours if the plant tissue is mounted in water (Chen and Heath 1991 ; H. Xu, unpublished data). Therefore, it is important to know whether the changes in [Ca2+]i seen in water-mounted tissue are the same as those in tissue in which the timing of the HR more closely resembles that in the uninjured plant. Because the addition of kinetin to the cellular bathing medium restores the timing of cell death to that seen in the intact plant (Chen and Heath 1991 ), we used kinetin-mounted tissue to obtain ratio values from three cells from each cowpea cultivar that exhibited cytoplasmic streaming and were uninfected, had only the fungus within the plant wall, or had a fungal intraepidermal vesicle in the cell lumen.
As shown in Table 2, ratio values for uninfected cells are much higher than those reported in Table 1 for comparable cells from water-mounted tissue. However, the changes in [Ca2+]i were similar to those seen in the latter tissue; values for resistant cells in which the fungus was only in the epidermal cell wall were higher than values for uninfected cells, with the highest values being recorded for cells in which the plant nucleus was moving to or was associated with the penetration site. A resistant cell with a small intraepidermal vesicle also had a high ratio value, but values were lower for cells containing larger intraepidermal vesicles. In susceptible cells, a particularly high value was observed for one cell in which the fungus was within the plant wall and the plant nucleus had migrated to the penetration site. However, when means of the values in each of the infection categories were compared with the mean for the comparable uninfected cells, only the means for resistant cells with the fungus in the plant wall were significantly different (Student's t test; P < 0.05).
To obtain more precise data on the relationship between the increase in [Ca2+]i in resistant cells and plant nuclear movement to the penetration site, ratio values for a single plant cell from kinetin-mounted tissue were recorded at 2- or 3-min intervals for a period of ~80 min. During this time, the nucleus migrated to the site of the fungus within the plant wall. Figure 5 shows some fluctuations in ratio values over this period, but a clear increase can been seen during the first few minutes of observation. Another increase occurred at the time the nucleus started to move to the penetration site.
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Figure 5.
Association of Increased [Ca 2+]i with Plant Nuclear Migration.
Images were recorded for a single resistant cell from tissue mounted in kinetin starting (time 0) at ~10 min after dye injection. The fungus was within the plant wall, and arrows mark the time at which the plant nucleus (N) started to move to and reached the penetration site (PS). Fluctuations in ratio values were seen throughout the observed period of 80 min.
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Collectively, these data show that a similar transient increase in [Ca2+]i occurs during fungal growth through the resistant plant wall in both kinetin-mounted tissue and water-mounted tissue in which the HR is delayed. Therefore, the timing of this [Ca2+]i increase is related to the stage of fungal development rather than to the time of the initiation of cell death.
Periodic Fluctuations in [Ca2+]i
The data shown in Figure 5 suggest that there might be periodic oscillations in [Ca2+]i, as has been reported during calcium-mediated signaling in other situations (Ehrhardt et al. 1996 ). Therefore, ratio values were taken from additional individual, representative cells at frequent intervals over a period of ~20 to 30 min. Figure 6B shows that in the susceptible plant, there was no detectable fluctuation in ratio values for an uninfected cell or for one with the fungal penetration peg in the plant wall. However, there was some fluctuation in cytoplasmic calcium levels in a cell containing a large intraepidermal vesicle. As shown in Figure 6A, resistant cells that were uninfected or contained a fungal intraepidermal vesicle did not show striking fluctuations in ratio values, but some fluctuations were recorded for the cell in which the fungus was still within the plant wall. It should be pointed out, however, that our experimental procedure made it difficult to obtain values at uniform or very small time intervals, and very rapid oscillations could have been missed.
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Figure 6.
Changes in [Ca2+]i Levels in Single Resistant or Susceptible Cells.
Representative curves showing the changes with time in ratio values from individual cells. The first observation (time 0) was made at ~5 to 10 min after injection of the dyes.
(A) Curves of ratio values for resistant cells, with the fungus within the plant wall and the plant nucleus beneath the penetration site (squares) or with the fungus in the plant cell lumen (triangles). The circles show the ratio values for an uninfected control cell.
(B) Curves of ratio values for susceptible cells, with the fungus within the cell wall and the plant nucleus near the penetration site (squares) or with the fungus in the plant cell lumen and the plant nucleus around the fungus (triangles). The circles show the ratio values for an uninfected cell.
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Effect of Calcium Channel Inhibitors on the Elevation of [Ca2+]i
Elevation of [Ca2+]i is caused by calcium influx from calcium stores through membrane calcium channels. In plants, both extracellular and intracellular stores are thought to be involved (Bush 1995 ). We investigated the effect on [Ca2+]i of two calcium channel inhibitors (lanthanum chloride, a plasma membrane channel blocker, and verapamil, a membrane channel blocker) by feeding them through the plant roots for 24 hr; injection into the intercellular spaces of the leaf, as was done during later experiments to study the HR in intact plants, was not effective because the inhibitors did not diffuse well into the vein epidermal cells in which [Ca2+]i was measured. Table 3 shows that ratio values of infected, resistant cells are similar for both early (fungal penetration peg within the epidermal wall) and later (small intraepidermal vesicle in plant cell) stages of infection and similar to values for untreated, uninfected plants (see Table 1). Therefore, the inhibitors had prevented the increase in [Ca2+]i normally seen in resistant cells during fungal growth in the plant wall.
Pharmacological Tests
We also tested whether the outcome of the fungus–plant interaction could be changed by chemical agents that affect Ca2+ metabolism, such as EGTA (an extracellular chelator), TMB-8 (an intracellular antagonist), LaCl3, and verapamil. As shown in Figure 7, injecting these chemicals into the apoplast of leaves of susceptible, intact plants had little or no effect on fungal infection with the exception of LaCl3, which induced cell death in ~10% of infected epidermal (non-vein) cells. In contrast, LaCl3, verapamil, and TMB-8 significantly delayed the HR in resistant plants (defined as any detectable stage in the death process, such as the granulation of plant cytoplasm or cell browning; Chen and Heath 1991 ). EGTA, which is not thought to enter cells but to chelate extracellular (apoplastic) calcium, predominantly affected fungal development (Figure 7A and Figure 7B), with ~80% of the basidiospore germlings halting their development at the appressorium stage.
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Figure 7.
Pharmacological Tests with Resistant and Susceptible Plants.
(A) and (B) Effect of calcium inhibitors on fungal development in susceptible (A) and resistant (B) plants. Basidiospore development was divided into five stages (Xu and Mendgen 1991 ), namely, ungerminated spore, germinated spore (with long or short germ tube), germ tube with appressorium (APP), fungal hypha in a still living plant cell (LIV) (i.e., showing no cytological signs of cell death), and fungal hypha in a cell showing signs of the death process (NEC). The frequencies of the first two stages, representing ~10% in all treatments, are not presented. Mean values (±SD) were obtained from at least 600 basidiospores and infection sites counted in six leaves from three separate trials.
(C) and (D) Mean hyphal length (±SD) in susceptible (C) and resistant (D) plant cells treated with calcium inhibitors. Values were calculated from at least 150 infection sites in three separate trials. Values for all inhibitor treatments in (D) recorded at 12, 16, and 24 hr after inoculation were significantly different from the comparable water treatment values at P < 0.05 (Student's t test).
W, water control; L, LaCl3; V, verapamil; T, TMB-8; E, EGTA; CB, susceptible cultivar California Blackeye; DC, resistant cultivar Dixie Cream.
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To determine whether the delay of the HR by calcium inhibitors was caused by an effect on the fungus, we measured hyphal lengths in plant cells pretreated with the three effective inhibitors. As summarized in Figure 7C and Figure 7D, mean hyphal length in the susceptible plant was not influenced by these inhibitors during the first 16 hr after inoculation, at which point 95% of the infected plant cells in the resistant plant showed signs of HR (data not shown). In resistant plants harvested at 12, 16, and 24 hr after inoculation, the three inhibitors significantly increased fungal growth compared with the water control.
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DISCUSSION |
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Accumulating evidence suggests that there are marked differences in the responses of plant cell cultures (including protoplasts) and those of intact plant tissue (Showalter et al. 1985 ; Bell et al. 1986 ; Lawton and Lamb 1987 ; Cuypers et al. 1988 ; Xiang et al. 1996 ). Nevertheless, most data on the role of calcium in the signal transduction leading to plant defense responses have been obtained from cell cultures treated with elicitors (Mahady and Beecher 1994 ; Messiaen and Van Cutsem 1994 ; Suzuki et al. 1995 ; Tavernier et al. 1995 ; Ishihara et al. 1996 ; Levine et al. 1996 ). Our study demonstrates the changes in cell calcium levels during the HR in a natural fungus–plant interaction.
Calcium is used by plant cells as a secondary messenger to control many cell processes, such as stomatal closure (Gilroy et al. 1991 ; Ward et al. 1995 ; Webb et al. 1996 ), tropism (Gehring et al. 1990 ; Williams et al. 1990 ; Sinclair et al. 1996 ), orientation of pollen tube growth (Malho and Trewavas 1996 ; Hepler 1997 ), cold acclimation (Knight et al. 1996 ), gene expression, and many calcium-dependent enzyme activities (Roberts and Harmon 1992 ). In plants, two kinds of calcium stores are believed to contribute to [Ca2+]i: extracellular (apoplastic) stores in the cell wall and intracellular stores in the vacuole and endoplasmic reticulum (Bush 1995 ).
In this study, we report that the HR induced by fungal infection in intact plants was delayed by the calcium channel inhibitors LaCl3, verapamil, and TMB-8. These inhibitors were used at concentrations similar to those used in other studies involving plant material (Gilroy et al. 1991 ; Zonia and Tupy 1995 ; Ishihara et al. 1996 ). LaCl3, a widely used calcium channel blocker, is believed not to penetrate the plasma membrane because of strong positive charges (Evans 1990 ). Therefore, LaCl3 would be expected to block calcium entry through the plasma membrane from the cell wall. In contrast, verapamil, a phenylalkylamine derivative, is an effective inhibitor of Ca2+ channels in a variety of membranes when applied to the intracellular surface of the channel, where it binds to the transmembrane pore and occludes it (Catteral and Striessnig 1992 ). TMB-8 is reported to prevent calcium release from intracellular stores in animal (Singh et al. 1994 ; Malcolm et al. 1996 ) and plant (Alexandre et al. 1990 ; Zonia and Tupy 1995 ; Ishihara et al. 1996 ) cells, although other cellular effects have been reported (e.g., Palmer et al. 1992 ). Our pharmacological results therefore suggest that the release of calcium into the cytoplasm from extracellular and possibly intracellular calcium stores is involved in the HR.
EGTA, a calcium chelator widely used in the study of calcium function, was not useful in our system. On leaves pretreated with EGTA, most basidiospore development was halted at the appressorium stage, which precedes the fungal entry into the cell. The necessity of calcium for fungal development has also been demonstrated for a powdery mildew pathogen (Takamatsu et al. 1978 ).
We visualized the change of [Ca2+]i in epidermal cells during fungal infection by using fluorescence ratio analysis after microinjection of dyes linked to the same dextran—CG-1 for reporting [Ca2+]i and Texas Red for reporting dye concentration. The cells loaded with the dyes were living, and cytoplasmic streaming occurred normally during the period of analysis. In resistant plants, cells loaded with the dyes eventually exhibited the HR (data not shown), indicating that dye injection did not significantly interfere with the plant response. Dye injection proved to be superior to acid or ester loading (Read et al. 1992 ) in our system. Acid loading induced cell death, and acetoxylmethyl ester loading was not successful (H. Xu, unpublished data); the latter result is probably due to acetoxylmethyl ester loading problems in plant tissue, as decribed by Cork 1986 .
Ratio analysis and imaging revealed a consistent increase in [Ca2+]i in resistant but not susceptible cowpea cells at the time when the fungus was penetrating the plant cell wall. This increase was observed in detached leaf tissue bathed in water, in which the HR can be delayed by several hours relative to the intact plant, as well as in tissue bathed in kinetin solution, in which the timing of the HR is normal. These observations suggest that the increase in [Ca2+]i is related to the stage of fungal development and not to the timing of the HR. Therefore, although high calcium levels are capable of initiating protein degradation (Sen 1996 ), it seems likely that the observed increase in [Ca2+]i was not directly responsible for hypersensitive cell death. This conclusion is supported by the decline in [Ca2+]i after the fungus began growing within the cell lumen.
Interestingly, bathing the cells in kinetin increased the baseline [Ca2+]i in uninfected plant cells, as has been shown for other growth regulators in other situations (Gilroy and Jones 1992 ; Schumaker and Gizinski 1993 ; Bhatla et al. 1996 ). Although this increase was higher in both susceptible and resistant cells than that induced by fungal infection in resistant plant cells bathed in water, it did not lead to cell death either in the absence of the fungus or when the fungus was invading susceptible cells. Therefore, a baseline elevation of [Ca2+]i per se is not sufficient to trigger the HR. However, the consistent increase in [Ca2+]i in the resistant fungal-infected cell coupled with the delay in cell death caused by calcium channel inhibitors and the demonstration that these inhibitors abolish the [Ca2+]i increase strongly support the hypothesis that in intact resistant plants, calcium is involved in the transduction of an HR-inducing signal.
During signal transduction in animal cells, elevation of [Ca2+]i tends to be fast and of short duration (Tsien and Tsien 1990 ). However, the elevation of [Ca2+]i that we observed here was slow and prolonged. Possibly, fungal products cause a dosage-dependent elevation of [Ca2+]i (Messiaen et al. 1993 ) and are released very slowly by the fungus or the fungus–plant interaction over a long period of time. Slow and prolonged elevation of [Ca2+]i has been reported in other situations in plants (Gilroy et al. 1991 ; Messiaen et al. 1993 ; Ehrhardt et al. 1996 ). Messiaen et al. 1993 suggested that this might provide a mechanism by which plants could easily reverse the effects of an extracellular signal(s) within a certain time period.
The periodicity of regular oscillations in [Ca2+]i, as opposed to sustained elevation, may be important in the outcome of a signaling event (discussed in Ehrhardt et al. 1996 ), which could explain the lack of cell death in kinetin-treated tissue with high baseline levels of [Ca2+]i. However, although fluctuations were observed in resistant, infected cells during our study, these fluctuations were not as consistent or regular as those reported for other systems (e.g., Ehrhardt et al. 1996 ), and their significance awaits further experimentation.
Penetration of the plant cell wall by the cowpea rust fungus is thought to be enzymatic (Mendgen and Deising 1993 ; Xu and Mendgen 1997 ). Enzymatic degradation products of cell walls could act as nonspecific elicitors of plant defense responses (Boller 1995 ; Hahn 1996 ). Such elicitors can increase [Ca2+]i in cell cultures or protoplasts (Messiaen et al. 1993 ; Mahady and Beecher 1994 ; Dmitriev et al. 1996 ) and may trigger the migration of the plant nucleus to the penetration site in both susceptible and resistant cowpea cells as the fungus grows through the cell wall (Heath et al. 1997 ). Nuclear migration is prevented by calcium chelators and the protein kinase inhibitor staurosporine, suggesting that the migration response is part of a nonspecific, calcium-mediated, and kinase-supported cascade initiated by pathogen invasion (Heath et al. 1997 ). However, in our study, we could not detect any prolonged increase of [Ca2+]i in susceptible plant cells at any infection stage, although occasional cells with high values were observed. Because nonspecific defensive responses to the fungus seem to be rapidly inhibited in both resistant and susceptible cells (Heath et al. 1997 ), the elevation of [Ca2+]i mediating nuclear migration may be too small or too transient to be detected by the method used in this study. In this context, it may be significant that the data shown in Figure 5 suggest that plant nuclear migration to the penetration site in the resistant cultivar coincides with the peak in the observed increase in [Ca2+]i.
The cowpea rust fungus–cowpea combination investigated here exhibits race cultivar specificity (Heath 1989 ; Chen and Heath 1991 ), as do many fungal–plant pathosystems. The greater and more sustained increase in [Ca2+]i observed in resistant cells in this work presumably results from specific interactions between the fungus and the resistant plant. Two fungal peptides that are produced when the fungus has formed an appressorium and that elicit cell death only in the resistant cowpea cultivar (Chen and Heath 1990 ) have been purified and sequenced (D'Silva and Heath 1997 ). Work is under way to develop an expression system to allow production of sufficient quantities of the elicitors to determine whether they are responsible for triggering the elevated [Ca2+]i seen in resistant cells.
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METHODS |
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Plant Materials and Fungal Isolate
The susceptible cultivar California Blackeye and the resistant cultivar Dixie Cream of cowpea (Vigna unguiculata) were grown in a growth chamber, as described previously (Heath 1989 ). Eight-day-old plants were used for confocal laser scanning microscopic measurements of cytosolic free calcium ([Ca2+]i), and 10-day-old plants were used for the pharmacological tests. Basidiospores of the cowpea rust fungus (Uromyces vignae) race 1 were obtained by incubating teliospores on 2% water agar in Petri dishes for ~2 days, as described previously (Chen and Heath 1991 ).
Chemicals
Two fluorescent dyes, Calcium Green-1 (CG-1) and Texas Red, linked to the same dextran (10 kD) were obtained from Molecular Probes, Inc. (Eugene, OR). CG-1 is a highly calcium-sensitive dye with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. It is widely used to report cell calcium level. Texas Red is calcium insensitive, and its excitation/emission wavelength is 596/615 nm.
Four calcium inhibitors, including three calcium channel blockers and a calcium chelator, were used as aqueous solutions in this study. LaCl3, which blocks plasma membrane calcium channels (Bush 1995 ), was obtained from Sigma and used at 1 mM (Gilroy et al. 1991 ) for injection of the inhibitor into the intercellular space of the leaf or 10 mM for feeding the plant with the inhibitor through the root (see below). Verapamil (Calbiochem, La Jolla, CA), a membrane calcium blocker, was used at 1 mM (Gelli and Blumwald 1993 ; Ishihara et al. 1996 ) for the injection or 0.5 mM for the feeding (see below). TMB-8 (8-[N,N-diethylamino]-octyl-3,4,5-trimethoxybenzoate, HCl; Calbiochem), an intracellular calcium antagonist, was used at 0.5 mM (Alexandre et al. 1990 ), and EGTA (Sigma), a calcium chelator, was used at 1 mM (Ishihara et al. 1996 ).
Kinetin (Sigma), an analog of the plant growth regulator cytokinin, was dissolved in water to form a saturated solution. Excess kinetin was removed by filtering.
Inhibitor Treatments
To investigate the effects of inhibitors on [Ca2+]i levels, 7-day-old plants were carefully removed from the soil, and their roots were washed with tap water. Approximately 1.5 cm of the root tips were cut off, and the roots were placed in 10 mM LaCl3 or 0.5 mM verapamil, at which inhibitor concentrations the inhibitory effect on the hypersensitive response (HR) in the main vein epidermal cells was similar to that seen in epidermal cells overlying the mesophyll during the pharmacological tests (see below). After 24 hr in the growth chamber, the main veins of the plant leaves were inoculated with the fungus. For the pharmacological tests, inhibitors were injected into the intercellular spaces of the leaf with a 1-mL syringe, without a needle, applied to the lower epidermis of 10-day-old plants. Control injection was with water. After injection, the leaf surface was washed with water, and the remaining water droplets on the leaf surface were wiped off with tissue. The injected, water-soaked leaf area was allowed to dry under laboratory conditions for 40 to 60 min before inoculation with the fungus.
Inoculation
Because of the asynchronous release of basidiospores from the germinated teliospores, plants were inoculated as described by Xu and Mendgen 1991 for the pharmacological experiments to achieve a relatively synchronous inoculation. The Petri dish containing germinating teliospores was turned upside down and placed over the plant leaf in high humidity. The release of basidiospores was allowed to occur for 4 hr. The Petri dish was then removed, and the inoculated plant was subsequently incubated in high humidity in the dark until harvest. For confocal laser scanning microscopy, a piece of agar bearing germinating teliospores was placed over the main leaf vein. The plant was then incubated in high humidity in the dark until use (Chen and Heath 1991 ).
Tissue Preparation for Confocal Laser Scanning Microscopy
The cytoplasm of fully expanded cowpea leaf vein epidermal cells (10 days old) is only ~0.4 µm thick, whereas the width of the cell wall is ~1 µm. In these cells, it is difficult to microinject dyes into such a thin layer of cytoplasm. Instead, dyes are often injected into the vacuole, which kills the cells. Therefore, microinjection and confocal laser scanning microscopy were performed with the half-expanded cells of 8-day-old plants, although younger plants showed less fungal resistance than did the older (10-day-old) ones (data not shown). Six to 10 hr after inoculation, the inoculated main vein, with ~1 cm of mesophyll tissue on either side, was cut from the leaf. The lower part of the main vein was sliced off with a razor blade, and the leaf piece was laid upper side up on a drop of double-distilled water on a microscope slide. For kinetin experiments, a similar leaf piece was first incubated in kinetin solution for ~30 min and then laid on a drop of the kinetin solution on a microscope slide. The leaf piece was clamped by putting a coverslip on each end to prevent the leaf piece from moving during the experiment. Each leaf piece usually was used for a maximum of 1 hr.
Microinjection of Dyes into Epidermal Cells and Confocal Laser Scanning Microscopy
Micropipettes were pulled from filament electrode glass by using a vertical pipette puller (model p-30; Sutter Instrument Co., San Rafael, CA). Micropipette tips were ~1 µm in diameter. The dye CG-1–Texas Red dextran (100 µM) was dissolved in micropore-filtered distilled water. The micropipette was back-filled with the dye solution of CG-1–Texas Red by capillary action. The remaining volume of the micropipette was thereafter filled with 100 mM KCl, with the exception of a small air space, which remained between the dye and KCl. Epidermal cells were injected by pressure injection with a 50-mL syringe. The injection time was ~10 to 20 sec. Injected cells were left for ~5 to 10 min for recovery and distribution of the dye throughout the cytoplasm.
Calcium imaging was performed with a BioRad MRC 600 confocal laser scanning microscope equipped with a x40, NA 0.8, dry objective and an IBM computer with CoMos software. Laser illuminations at 488 and 568 nm (krypton/argon) were dually recorded through a 515- to 540-nm or 589- to 621-nm bandpass filter for CG-1 and Texas Red, respectively. Several dual images of the same cell were collected, usually at random intervals, and stored on an optical diskette (Verbatim Corp., Charlotte, NC). Prints from digital images were created using a Sony video graphic printer (model Up-860).
Ratio Analysis
Numerical ratio analysis was performed using Histogram/CoMos software supplied within the IBM computer by comparing the average number of photons per pixel in a comparable boxed area for each pair of CG-1 and Texas Red images. The average ratio value of several recorded dual images of a single cell was regarded as the ratio value of the cell. Visual images of pixel-by-pixel ratios for entire cells were made using an Image-1 image processing and analysis system (Universal Imaging Corporation, West Chester, PA) and printed using a video copy-processor (model P4OU; Mitsubishi Electronic America, Inc., Somerset, NJ).
A calcium calibration kit with magnesium (C-3721, No.1; Molecular Probes) was used to calibrate ratio values of CG-1 to Texas Red versus free Ca2+ concentrations in vitro. To avoid calcium leaching from glassware, plastic microscope slides were made from polystyrene Petri dishes (Fisher Scientific Co., Whitby, Ontario, Canada), and the trademark impression was used as a microwell. Glass coverslips were coated with plastic wrap. Slides and coverslips were washed extensively with double-deionized water and dried at 60°C. Approximately 2 µL of the appropriate free Ca2+ buffer solution containing 10 µM CG-1–Texas Red dye was added to the microwell and covered with a coverslip. Calcium imaging with the confocal laser scanning microscope and ratio imaging analyses were performed as described above.
Tissue Preparation for Light Microscopy
After harvest, the leaf pieces were fixed either in 2% glutaraldehyde in 50 mM phosphate buffer, pH 7.6, for 30 min or in 95% ethanol for several hours at room temperature. The leaf pieces fixed in glutaraldehyde were rinsed in tap water twice and then gently vacuum infiltrated with tap water (Xu and Mendgen 1991 ) and mounted in tap water. The ethanol-decolorized leaf pieces were hydrated in tap water for 10 min and mounted in 30% (v/v) glycerol and water. Microscopy was performed using a Reichert-Jung Polyvar microscope (Reichert AG, Vienna, Austria) equipped with differential interference contrast optics.
Data were collected from infected epidermal cells throughout the leaf surface. Because the hyphae in plant cells are rarely vertical or horizontal, the proportional equation C2 = A2 + B2 was used to accurately measure hyphal lengths, where C is the hyphal length, A is the horizontal length measured by an eyepiece micrometer, and B is the vertical length measured by the fine focus control of the microscope. The fine focus control was calibrated with glass beads. Any cell showing any cytological signs of the death process (Chen and Heath 1991 ) was considered to be exhibiting an HR.
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ACKNOWLEDGMENTS |
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We thank John D.W. Dearnaley and Mary-Lou Ashton (York University, Toronto, Ontario, Canada) for assistance with confocal microscopy and image analysis and Eduardo Blumwald (University of Toronto, Canada) for discussions. This work was funded by the Natural Sciences and Engineering Research Council of Canada.
Received December 3, 1997; accepted February 13, 1998.
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INTRODUCTION
