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First published online October 22, 2004; 10.1104/pp.104.049650 Plant Physiology 136:3703-3711 (2004) © 2004 American Society of Plant Biologists Oxalate Production by Sclerotinia sclerotiorum Deregulates Guard Cells during Infection1,[w]Department of Horticulture, Oregon State University, Corvallis, Oregon 97331
Oxalic acid is a virulence factor of several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, but the detailed mechanisms by which oxalic acid affects host cells and tissues are not understood. We tested the hypothesis that oxalate induces foliar wilting during fungal infection by manipulating guard cells. Unlike uninfected leaves, stomatal pores of Vicia faba leaves infected with S. sclerotiorum are open at night. This cellular response appears to be dependent on oxalic acid because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. In contrast to oxalate-deficient S. sclerotiorum, wild-type fungus causes an increase in stomatal conductance and transpiration as well as a decrease in plant biomass. Green fluorescent protein-tagged S. sclerotiorum emerges through open stomata from the uninfected abaxial leaf surface for secondary colonization. Exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening. Guard cells treated with oxalic acid accumulate potassium and break down starch, both of which are known to contribute to stomatal opening. Oxalate interferes with abscisic acid (ABA)-induced stomatal closure. The Arabidopsis (Arabidopsis thaliana) L. Heynh. mutants abi1, abi3, abi4, and aba2 are more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. We conclude that oxalate acts via (1) accumulation of osmotically active molecules to induce stomatal opening and (2) inhibition of ABA-induced stomatal closure.
Oxalic acid (ethanedioic acid) occurs ubiquitously in nature, sometimes as a free acid, but more commonly as soluble potassium or sodium oxalate or as insoluble calcium oxalate. Biosynthesis of oxalate occurs in members of all five kingdoms. Oxalate is associated with metabolic disorders and infectious diseases (Holmes and Assimos, 1998  ; Nakagawa et al., 1999). Several phytopathogenic fungi, including Sclerotinia sclerotiorum (Lib.) de Bary, produce millimolar concentrations of oxalate in infected tissues (de Bary, 1886; Ferrar and Walker, 1993). Oxalate is an essential virulence factor of S. sclerotiorum because mutants, which are deficient in oxalate biosynthesis, are less pathogenic than wild-type fungus (Godoy et al., 1990). In contrast to wild-type fungus, oxalate-deficient S. sclerotiorum is unable to produce oxalate during infection of petals, which are an important source of inoculum in the field and during in vitro cultivation (Godoy et al., 1990; Jamaux et al., 1995).
Enzymes that catabolize oxalate protect plants from Sclerotinia infection when their genes are expressed in stably transformed plants. Constitutive expression of wheat oxalate oxidase, an enzyme that converts oxalate into H2O2 and CO2, enhances resistance of soybean (Glycine max; Donaldson et al., 2001
The precise mechanism of oxalate action during infection is not completely understood. However, oxalate has been proposed to remove calcium ions bound to pectins, which exposes host cell walls to catabolic enzymes of fungal origin (Bateman and Beer, 1965
Oxalic acid causes wilting symptoms in Sclerotinia-infected plants (Noyes and Hancock, 1981
S. sclerotiorum Induces Oxalate-Dependent Wilting Symptoms by Deregulating Guard Cells
We used a green fluorescent protein (GFP)-tagged strain of S. sclerotiorum in conjunction with confocal microscopy to determine whether wilting symptoms (Fig. 1A) are the result of stomatal dysfunction during infection of Vicia faba leaves. S. sclerotiorum prevented closure of stomata in the dark (Fig. 1, B–D). The fungus exploited open stomatal pores to emerge from the uninoculated abaxial leaf surface (Fig. 1C; Supplemental Fig. 1, available at www.plantphysiol.org). Based on microscopic analysis of four leaves from two plants 2 d postinoculation (dpi), 22 ± 1 hyphae protruded through stomata, whereas 7 ± 1 hyphae penetrated through the cuticle (paired t test; n = 118; P = 0.0008). Stomata were open in advance of fungal colonization (Fig. 1B). All of the stomatal pores in the vicinity of hyphal growth were classified as open (
To determine whether stomatal dysfunction depends on the production of oxalic acid, we compared stomatal apertures after infection of V. faba leaves with wild-type or oxalate-deficient S. sclerotiorum. Oxalate deficiency was confirmed by comparing oxalate accumulation in leaves infected with wild-type or mutant fungus (Fig. 2A). In contrast to wild-type S. sclerotiorum, oxalate levels in leaves infected with mutant fungus were not significantly different from control leaves. Two types of control leaves were used: (1) leaves removed prior to the onset of the experiment and (2) leaves mock-inoculated with agar and harvested 2 dpi. Oxalate concentrations increased 38-fold in leaves infected with wild-type S. sclerotiorum 2 dpi. Unlike wild-type S. sclerotiorum, which caused soft rotting lesions, mutant fungus caused a dry necrosis (Fig. 2B). These results are consistent with published data (Godoy et al., 1990 ) and suggest that differences in stomatal responses are likely due to defects in fungal oxalate biosynthesis. The oxalate-deficient mutant was less virulent than the wild-type fungus (Fig. 2B). Changes in stomatal aperture were detected within and around necrotic lesions. Open stomata occurred 5 mm beyond the necrosis in leaves infected with wild-type S. sclerotiorum, but only 0.8 mm beyond the necrosis in leaves infected with oxalate-deficient fungus. Disease susceptibility and stomatal aperture were closely correlated. Stomatal apertures of leaves infected with wild-type fungus were twice as wide in necrotic areas and five times wider in areas surrounding necroses when compared to leaves infected with the mutant fungus (Fig. 3).
We determined the effect of oxalate-dependent stomatal dysfunction on whole-plant physiology. Necrotic lesions advanced four times faster in stems of V. faba plants infected with wild-type S. sclerotiorum relative to the oxalate-deficient mutant fungus (Table I). Stomatal conductance and transpiration rates were significantly higher in plants infected with wild-type fungus than in plants challenged with the oxalate-deficient mutant or mock-inoculated plants. In addition, wild-type S. sclerotiorum significantly reduced stem fresh weight and dry weight over a period of 12 d when compared to the oxalate-deficient mutant or mock-inoculated controls. These data suggest that S. sclerotiorum manipulates stomata to increase water stress and pierce through the abaxial leaf surface.
Oxalate Induces Stomatal Opening in Detached Leaf Epidermis
We treated the abaxial epidermis of V. faba leaves with oxalic acid to characterize guard cell behavior. Oxalate induced maximal stomatal opening at a concentration of 1 and 10 mM oxalate at pH 3 and 6.3, respectively (Fig. 4A). Because the pKa values of oxalic acid are 1.2 and 4.2, the majority of this acid is expected to exist as monodissociated and completely dissociated forms at pH 3 and 6.3, respectively. The aperture of stomatal pores increased with time (Fig. 4B). Concentrations of 1 to 10 mM oxalate significantly increased stomatal opening relative to the buffer control at pH 6.1 over a period of 3 h. Fluorescein diacetate staining indicated that loss of cell viability at 10 mM oxalate, pH 3, and at 100 mM oxalate, pH 6.3, caused a decline in stomatal aperture (Fig. 4). No reduction in cell viability was observed at
We tested the dicarboxylic anions oxalate, malate, malonate, and succinate for specificity of stomatal responses at 1 mM, pH 6.1. Oxalate was the only dicarboxylic anion that significantly increased stomatal aperture (Table II). Thus, guard cells specifically respond to oxalate in a concentration and pH-dependent manner.
Oxalate Stimulates K+ Uptake and Starch Degradation in Guard Cells To determine whether oxalate-induced changes in stomatal aperture are caused by solute accumulation, we measured potassium uptake and starch degradation in guard cells of detached abaxial leaf epidermis. Oxalic acid treatment significantly increased uptake of K+ into guard cells compared to the buffer control (Fig. 5, A, B, and D). The staining pattern after oxalate or fusicoccin treatment was similar (Fig. 5, B and C). However, the amount of K+ uptake into guard cells was significantly lower in the case of oxalate-treated epidermis compared to fusicoccin (Fig. 5D). Because oxalate may alter the content of organic solutes in guard cells, we also measured starch content. Oxalate significantly decreased the amount of starch in guard cell chloroplasts (Fig. 5, E–G).
Because K+ uptake and starch degradation suggest alterations in osmotic pressure, we isolated guard cell protoplasts and exposed them to 10 mM oxalic acid. The volume of these protoplasts increased significantly in the presence of oxalate (Fig. 6, A and B), suggesting that increases in cellular solutes are responsible for stomatal opening. We also observed a significant decrease in cell number (Fig. 6C) because oxalate-treated cells burst and die. Collectively, these results suggest that an increase in osmotically active solutes is responsible for oxalate-dependent stomatal opening.
Interaction between Oxalate and ABA: Its Effect on Stomatal Control and Susceptibility to S. sclerotiorum Because oxalate produced by S. sclerotiorum interferes with stomatal closing at night, we reasoned that oxalate may also prevent ABA-induced stomatal closure. To test this hypothesis, we challenged the abaxial epidermis of V. faba with different concentrations of oxalate in the presence or absence of 100 µM ABA. The aperture of light-adapted stomata decreased in response to ABA (Fig. 7). This ABA-induced decrease in stomatal aperture was significantly reduced by cotreatment with 1 and 10 mM oxalate. Oxalate significantly increased stomatal aperture compared to the buffer control at a concentration of 10 mM (paired t test; P < 0.001), but not at 1 mM (paired t test; P = 0.87).
If stomatal aperture plays a significant role in S. sclerotiorum virulence, mutants deficient in stomatal closure and ABA signaling would be expected to have increased susceptibility to fungal infection. We used the ABA-insensitive mutants abi1 and abi2, which are impaired in stomatal closure, causing increased leaf transpiration and wilting (Leung et al., 1994 , 1997). Each of these mutants has a defect in a different, but homologous, protein phosphatase 2C that acts downstream of ABA perception (Leung et al., 1994, 1997; Murata et al., 2001). The mutants abi3 and abi4 have defects in ABA-dependent transcriptional activators without known effects on guard cell regulation (Koornneef et al., 1984; Soderman et al., 2000). The aba2 mutant is impaired in ABA biosynthesis and stomatal regulation and sensitive to water stress (Schwartz et al., 1997; Merlot et al., 2002). We challenged these mutants and their wild-type backgrounds with the oxalate-deficient mutant of S. sclerotiorum to avoid the confounding influence of oxalate on disease development. The abi4 and aba2 mutants were significantly more susceptible to fungal attack than wild-type Arabidopsis (Arabidopsis thaliana) L. Heynh. ecotype Columbia (Col-0; Fig. 8A). The abi1 and abi3 mutants were more susceptible to fungal infection than wild-type Arabidopsis ecotype Landsberg (Ler-0; Fig. 8B). However, the abi2 mutant was not significantly more susceptible than its corresponding ecotype Ler. Collectively, these results suggest that ABA contributes to resistance against S. sclerotiorum apparently by antagonistic interaction with oxalate.
In this article, we present evidence for guard cell dysfunction during infection of V. faba with S. sclerotiorum. This pathogen inhibits closure of stomatal pores in the dark via an oxalic acid-dependent mechanism because this cellular response was partially suppressed when plants were exposed to an oxalate-deficient fungal mutant. We confirmed oxalate deficiency by measuring oxalate production in leaves of infected V. faba plants. Oxalate concentrations were 25 times higher in leaves infected with wild-type S. sclerotiorum than in leaves challenged with the oxalate-deficient mutant 2 dpi. Whereas the mutant fungus did not significantly increase foliar oxalate levels, oxalate concentrations of approximately 10 mM were measured 2 dpi when leaves were challenged with wild-type S. sclerotiorum. Accumulation of oxalate in infected tissues, therefore, reaches concentrations (de Bary, 1886 ; Ferrar and Walker, 1993) that are high enough to induce stomatal opening in vitro. Supplementation of the fungal growth medium with succinate, which is an inducer of oxalate production, enhanced lesion expansion, albeit insignificantly (Supplemental Fig. 2). This marginal increase in pathogenicity is consistent with other small, but significant, effects of succinate on virulence (Godoy et al., 1990; T.J. Chipps, B. Gilmore, J.R. Myers, and H.U. Stotz, unpublished data). Oxalate-deficient mutants, grown in the presence of succinate, still produce 5 times less oxalate than wild-type S. sclerotiorum (Godoy et al., 1990). This lower level of oxalate production is perhaps insufficient for the mutant to cause a dramatic shift in pathogenicity (Godoy et al., 1990; T.J. Chipps, B. Gilmore, J.R. Myers, and H.U. Stotz, unpublished data). Oxalate-deficient mutants grow 19% to 28% slower than wild-type S. sclerotiorum and do not produce sclerotia (Godoy et al., 1990). A reversion restored oxalate production, sclerotia formation, and growth rate, suggesting that all three phenotypes were caused by disruption of a single gene (Godoy et al., 1990). We factored out differences in growth rate when comparing stomatal apertures between wild-type and mutant S. sclerotiorum because we measured stomatal apertures within and outside necrotic lesions regardless of their size. Growth rate was therefore not relevant for these measurements. Thus, oxalate deficiency is most likely responsible for reduced stomatal responses to S. sclerotiorum, although we cannot entirely exclude the contribution of other factors because oxalate-deficient mutants have not been genetically characterized.
We detected open stomata in advance of invading hyphae, suggesting that oxalate moves faster than the mycelium. This observation supports previous data on the systemic spread of oxalate through the vascular tissue (Noyes and Hancock, 1981
In addition to causing water stress, open stomata were exploited for hyphal emergence and secondary colonization. We confirmed that initial penetration of V. faba leaves occurred via infection cushions (Prior and Owen, 1963
To understand the mechanisms of oxalate-dependent guard cell dysfunction, we studied stomatal responses in the detached abaxial epidermis of V. faba. Oxalate induces stomatal opening at concentrations between 1 and 10 mM. Because these concentrations are frequently exceeded in infected tissues (de Bary, 1886
Oxalate increased K+ uptake and starch degradation in guard cells. Potassium channels are known to be involved with osmosensing and turgor regulation of guard cells (Liu and Luan, 1998
Because secretion of oxalate by S. sclerotiorum prevents stomatal closure in the dark, we tested whether oxalate interferes with ABA-induced stomatal closing. Simultaneous exposure of the abaxial epidermis of V. faba to oxalate and ABA inhibited stomatal closing relative to treatment with ABA alone. At present, we do not know the mechanism by which oxalate suppresses ABA-induced stomatal closure. Oxalate may simply oppose ABA action by replenishing guard cells with K+. Alternatively, oxalate may modulate signal transduction processes. ABA is known to activate plasma membrane-localized Ca2+ and anion channels, leading to inactivation and activation of inward- and outward-rectifying K+ channels, respectively (Schroeder et al., 1987
The Arabidopsis mutants abi1, abi3, abi4, and aba2, which have defects in ABA sensing or ABA biosynthesis, were consistently more susceptible to oxalate-deficient S. sclerotiorum than wild-type plants. Because abi1 and aba2 mutants are impaired in guard cell regulation, stomatal opening is apparently required for optimal fungal colonization. In contrast to the abi1 mutant, increased susceptibility of the abi2 mutant to S. sclerotiorum was insignificant. This phenotypic difference may be due to the stronger ABA insensitivity of abi1 mutants relative to abi2 mutants (Leung et al., 1997
Regulation of stomatal movement involves a myriad of signals in response to an array of environmental and physiological cues (Hetherington and Woodward, 2003
Plant Material Arabidopsis (Arabidopsis thaliana) L. Heynh. mutant seeds aba2-1 (CS156), abi1-1 (CS22), abi2-1 (CS23), abi3-1 (CS24), and abi4-1 (CS3836) and ecotypes Col-0 and Ler-0 were obtained from The Arabidopsis Information Resource (TAIR). Vicia faba L. cv Broad Windsor seeds were purchased from Territorial Seed (Cottage Grove, OR). V. faba and Arabidopsis were cultivated from seeds under controlled environmental conditions. V. faba was grown with an 8-h photoperiod of white light (300 µmol m–2 s–1) at 25°C. Arabidopsis was grown with a 12-h photoperiod of white light (200 µmol m–2 s–1) at 25°C.
A wild-type isolate (1980) and an oxalate-deficient mutant (A4) of Sclerotinia sclerotiorum (Lib.) de Bary were kindly provided by Dr. Martin Dickman (University of Nebraska, Lincoln, NE) and grown on potato dextrose agar as described by Godoy et al. (1990)
To test effects on physiology of V. faba plants, we inoculated wounded stems of 1-month-old plants with agar containing wild-type or mutant S. sclerotiorum or agar alone (Petzholdt and Dickson, 1996
Leaves of V. faba plants were inoculated and incubated for up to 2 d with agar plugs containing S. sclerotiorum mycelia. Control leaves were mock inoculated with agar plugs and incubated for 2 d. Treated or control leaves (less than 0.8 g of fresh weight) were homogenized in 3.5 mL of 0.2 M potassium phosphate, pH 6.5 (Ferrar and Walker, 1993
The abaxial epidermis was peeled from the youngest fully expanded leaves of 1-month-old plants of V. faba. Epidermis was floated on a bath solution containing 45 mM KCl, 5 mM KOH, and 10 mM MES, pH 6.1, with or without treatment; pH was adjusted with NaOH. Opening experiments were initiated with closed stomata from leaves kept in darkness. Closure experiments were initiated with open stomata from leaves kept in white light (34 µmol m–2 s–1) at 20°C for 2 h. Epidermis was incubated in the bath solution for 1 h prior to treatment. Width of the innermost cell wall of guard cells was determined with an ocular micrometer and a Leica (Wetzlar, Germany) DME light microscope.
A GFP-tagged strain of S. sclerotiorum (Lorang et al., 2001
Fluorescein diacetate was used at a concentration of 0.01% to assess cell viability as recommended previously (Poffenroth et al., 1992
Epidermis was treated with or without 10 mM oxalic acid in bath solution containing 45 mM KCl, 5 mM KOH, and 10 mM MES, pH 6.1, for 2 h at 20°C in the dark, washed in water, and stained with 10% Lugol's iodine solution (ICN, Costa Mesa, CA) for 10 min. Epidermis was briefly rinsed with water prior to microscopy and image analysis. Cell walls were erased from guard cell images using Photoshop (Microsoft, Seattle) and the stained areas of chloroplasts were assessed. All images were converted to 8-bit using Image Pro Plus and analyzed using a range of 0 and 90. Cumulative data were exported and analyzed using Excel (Microsoft).
Sodium hexanitrocobaltate staining was used to measure K+ content; fresh solutions were prepared prior to each experiment (Green et al., 1990
To measure changes in osmotic pressure, we isolated guard cell protoplasts from V. faba according to Pandey et al. (2002)
Statistical tests included ANOVA, using the SAS program package (Cary, NC). Differences in stomatal conductance, transpiration, lesion size, fresh weight, and dry weight between treatments were compared using Duncan's Multiple Range Test. Student's t tests were used to compare treatment and control means for experiments involving detached epidermis. Experiments with Arabidopsis mutants and ecotypes were compared using the repeated-measures procedure of a general linear model; means were compared using LSD.
The authors are grateful to Dr. Jim Myers for assisting with statistical analysis and critical review of the manuscript; thanks also to Dr. Gary Tallman and Dr. Lawrence Talbott for valuable suggestions. Received July 13, 2004; returned for revision August 23, 2004; accepted August 24, 2004.
1 This work was supported by the U.S. Department of Agriculture Sclerotinia Initiative Special Grants program (grant no. 58–5442–2–257) and by the Department of Horticulture at Oregon State University. 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.049650. * Corresponding author; e-mail stotzhe{at}science.oregonstate.edu; fax 541–737–3479.
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5 µm; n = 50), whereas unchallenged leaves contained exclusively closed stomata (
5 µm; n = 50). 
) or oxalate-deficient mutant A4 (
; Godoy et al., 1990
). A mock-inoculated leaf was removed at the end of the experiment to test whether oxalate levels were altered during the course of the experiment (
). Means and SD of three plants are shown. B, Image of a plant 2 dpi. Compared to the wild-type strain, oxalate-deficient S. sclerotiorum was much less virulent.
-amino-butyric acid-induced resistance against the necrotrophic pathogen Plectosphaerella cucumerina (Ton and Mauch-Mani, 2004
