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Applied and Environmental Microbiology, September 2007, p. 5919-5927, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.02843-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae ^,

V. Cheng,¹ H. U. Stotz,³ K. Hippchen,² and A. T. Bakalinsky^1*

Department of Food Science and Technology, Wiegand Hall, Oregon State University, Corvallis, Oregon 97331-6602,¹ Environmental and Molecular Toxicology, Agricultural and Life Sciences Building, Oregon State University, Corvallis, Oregon,² Department of Horticulture, Agricultural and Life Sciences Building, Oregon State University, Corvallis, Oregon³

Received 6 December 2006/ Accepted 4 July 2007

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Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1, ric1, snf7, vps16, vps20, and vps51 mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2, vps16, vps51, ric1, and rib4 mutants, was lethal. With the exception of the rib4 mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4 mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.

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Oxalic acid (ethanedioic acid) occurs ubiquitously in nature (5). Multiple members of all five kingdoms (Monera, Protista, Fungi, Plantae, and Animalia) are able to produce oxalate (26). Oxalate overproduction or overexposure is associated with human ailments, such as kidney stone disease (13) and, in rare instances, pulmonary oxalosis (25). In fungi, oxalate serves ecological and pathogenesis-related functions. The availability of mineral nutrients increases when microbes secrete oxalate into the soil (10). Oxalate secretion also serves as a mechanism for detoxifying copper compounds (36). Secretion of oxalate by wood-rotting basidiomycetes appears to promote breakdown of lignin and cellulose (6, 29). Oxalic acid is a recognized virulence factor produced by several phytopathogenic fungi, including Sclerotinia sclerotiorum, the causal agent of white mold and related diseases (8). As such, oxalate likely mediates host cell wall degradation and plant disease symptoms by chelating pectin-bound calcium, by lowering the pH of infected plant tissue to a level more optimal for fungal cell wall-degrading enzymes, such as polygalacturonase (3), and by suppressing the defense-related oxidative burst (4). It was recently demonstrated that oxalate induces stomatal opening (11), which is at least partially responsible for wilting symptoms associated with white mold infections (27, 34).

Microbes and plants can catabolize oxalate via oxalate decarboxylase (15) and oxalate oxidase (19), respectively. Constitutive overexpression of both of these oxalate-degrading enzymes in plants has been shown to increase resistance to S. sclerotiorum (15). Overexpression of oxalate oxidase has been shown to increase expression of host defense genes, likely through the salicylic acid signaling pathway (14).

Saccharomyces cerevisiae is not known to produce or to catabolize oxalic acid. Baldwin (2) presented inconsistent data associating oxalic acid formation with growth of baker's yeast on a sugar-beef extract medium but declined to conclude that baker's yeast produced the oxalic acid. Nonetheless, it is likely that S. cerevisiae is exposed to oxalate in the soil environment (5, 33). While oxalate does not appear to be a normal metabolite of S. cerevisiae, the molecular targets for oxalic acid-mediated toxicity in this species may still be shared among plants and their oxalate-secreting fungal pathogens. We report herein on a genetic screen of a S. cerevisiae deletion library for mutants sensitive to oxalic acid, undertaken to discover oxalate-protective genes whose orthologs may encode protective functions in plants and in oxalate-secreting phytopathogenic fungi.

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Yeast strains and media.
The S. cerevisiae deletion library YSC1054Y, constructed with parent strain BY4742 MAT his31 leu20 lys20 ura30 or BY4739 MAT leu20 lys20 ura30, consists of deletions of nonessential genes (38) (Open Biosystems, Huntsville, AL). Cells were grown at 30°C in YEPD (1% yeast extract, 2% peptone, and 2% glucose), YEPD (pH 3.0), which is YEPD adjusted to pH 3.0 with HCl, or YEPD (pH 3.0) containing oxalic, malonic, formic, acetic, or propionic acid at specified concentrations. Liquid YEPD and YEPD agar were sterilized by autoclaving. All other liquid media were sterilized by filtration through a 0.45-µm filter.

Preliminary screens for oxalic acid-sensitive mutants.
Strains were grown in 200 µl of YEPD per well in 96-well microtiter plates for 24 to 48 h at 30°C (wrapped in parafilm to minimize evaporation) and were replica plated to wells containing 200 µl of sterile water per well using a 48-prong replicator as a means of diluting the inoculum. Cells were then replica plated from the water-containing plates to the test plates containing YEPD, pH 3.0, plus 30 mM oxalic acid, which were incubated wrapped in parafilm at 30°C. Growth was monitored daily and scored visually for turbidity after 5 days. The inoculum, which did not produce visible turbidity, ranged from 2,300 to 9,000 viable cells per well for 4 arbitrarily chosen mutants based on plating of a known dilution and volume on YEPD agar. Presumptive mutants that failed to grow or which grew poorly were retested at least once and were also tested in control medium, YEPD, pH 3.0. Mutants unable to grow in the control medium were eliminated from further consideration. Initially, pilot screens were undertaken with a limited number of deletion mutants to determine the appropriate oxalate concentration, pH, and inoculum size. The pH of the test medium was held constant at 3.0, since the toxicity of organic acids is pH dependent. MICs of oxalic acid and of the other acids indicated below were defined as those which resulted in slight or no turbidity (assessed visually) after 5 days of incubation relative to turbidity in the control medium, YEPD, pH 3. A total of 105 putative mutants were obtained in this preliminary screen and were subsequently tested for coincident sensitivity to formic, acetic, propionic, and malonic acids in YEPD, pH 3.0, using the protocol described above for the initial screen for oxalic acid-sensitive mutants, except that a number of concentrations of these other carboxylic acids were tested. For the list of 105 putative mutants, see Table S1 in the supplemental material.

To determine growth rates, overnight YEPD cultures of the 105 mutants were grown in 96-well microtiter plates at 30°C and replicated to YEPD (pH 3) and YEPD (pH 3) plus 30 mM oxalate in duplicate 96-well plates containing 200 µl of medium per well. Plates were wrapped with parafilm to minimize evaporation and incubated statically at 30°C. A₆₀₀ readings were taken hourly over 17 h in a microtiter plate reader (VERSAmax microplate reader; Molecular Devices) following plate mixing and reversible removal of the lids. Although the growth data generated were not reproducible due to apparent poor mixing of samples and excessive evaporative loss of medium, cell yield differences at the end of the 17-h time course between the control and oxalate-containing medium appeared to be substantial for about half of the 105 mutants. Therefore, these growth rate data were not used, but cell yields were subsequently determined quantitatively for the parent strains and for the 50 mutants that appeared to exhibit reduced growth in the oxalate medium. Growth rates were also determined quantitatively for four chosen mutants and their parents (secondary screens below).

The parent strains and 105 putative mutants were also tested for survival following exposure to oxalate. Strains grown in 200 µl of YEPD per well in 96-well microtiter plates for 24 to 48 h at 30°C (wrapped in parafilm to minimize evaporation) were replica plated in duplicate to 96-well plates containing YEPD (pH 3) and YEPD (pH 3) plus 30 mM oxalic acid, which were incubated for 3 days at 30°C. After the 3-day incubation, cells were transferred to YEPD agar using a 48-prong replicator. Growth on the YEPD agar plates was assessed by visual observation after 2 days at 30°C. The seven mutants that appeared to exhibit poorer survival in the oxalate medium relative to YEPD (pH 3) were subsequently retested under more rigorous conditions (secondary screens for sensitivity to oxalic acid).

Secondary screens for sensitivity to oxalic acid.
Cell yields of the parent strains and approximately 50 mutants that exhibited reduced visual turbidity in the preliminary screen for growth in oxalate medium were retested. Cultures were grown overnight in YEPD (pH 3) and diluted approximately 200-fold into 500 µl duplicate aliquots of YEPD (pH 3) and YEPD (pH 3) plus 30 mM oxalate. A₆₀₀ values of 10-fold-diluted cultures were measured following 3 days of static incubation at 30°C. The significance of differences in cell yields between YEPD (pH 3) and YEPD (pH 3) plus 30 mM oxalate was assessed at a P value of 0.05 by use of Student's t test. F statistics were used to determine homogeneity of variances prior to t tests.

Growth rates of four chosen mutants, the ada2, hom6, ptc1, and snq2 mutants, and their parent, BY4742, were determined. Cultures were grown overnight in an incubator-shaker at 30°C in YEPD (pH 3) and diluted to an A₆₀₀ value of about 0.1 in duplicate 1-ml aliquots of YEPD (pH 3) or YEPD (pH 3) plus 30 mM oxalate in sterile, capped, disposable 1.5-ml spectrophotometric cuvettes. Cultures were grown in an incubator-shaker at 30°C, and A₆₀₀ values were measured over approximately 7 h, sufficient time for determining growth rates. The statistical significance of differences was assessed by analysis of variance (SAS 9.1; SAS, Cary, NC).

The seven mutants that exhibited poorer survival in the presence of 30 mM oxalate than in the oxalate-free control medium in the preliminary screen were retested for survival under more rigorous conditions. Overnight cultures grown in an incubator-shaker at 30°C in YEPD (pH 3) were diluted approximately 200-fold into 500-µl aliquots of YEPD (pH 3), YEPD (pH 3) plus 1 mM oxalate, YEPD (pH 3) plus 5 mM oxalate, or YEPD (pH 3) plus 30 mM oxalate and incubated statically at 30°C. After 3 days of incubation, cultures were mixed well and 4-µl aliquots of undiluted or 10-fold-diluted cultures were spotted in duplicate on YEPD agar plates. These plates were then scored for growth after 2 days at 30°C. The five mutants among the seven tested that exhibited substantial loss of viability in the presence of 30 mM oxalate were subsequently tested for survival in the presence of 1, 5, or 30 mM acetate, propionate, malonate, and formate using the same protocol. Mutants that exhibited a complete lack of growth in both replicates of undiluted 4-µl aliquots were scored as sensitive. All plates contained the BY4742 parent strain as a positive control, which exhibited confluent growth following incubation in YEPD (pH 3) and in all oxalate-containing media. Aliquots from all mutants incubated in the YEPD (pH 3) control medium also exhibited confluent growth on YEPD agar.

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Putative oxalate mutants.
A total of 105 putative mutants were identified that grew in the control medium but failed to grow or grew poorly in the test medium, YEPD plus 30 mM oxalic acid (pH 3.0), as assessed visually. Table S1 in the supplemental material lists the deleted genes alphabetically by open reading frame (ORF) within biological process categories (http://db.yeastgenome.org/cgi-bin/GO/goTermMapper). Genes known to be involved in multiple processes are listed in multiple categories. MICs for oxalic, malonic, formic, acetic, and propionic acids are listed. Also indicated is whether the mutant was previously identified in published screens for sensitivity to sorbic (22, 30) or citric (20) acid. Gene functions are indicated if known, as are Arabidopsis thaliana and S. sclerotiorum orthologs and their respective probability scores. As of this writing, the S. sclerotiorum genome has not yet been fully annotated or published. The 28 entries listed in bold in Table S1 in the supplemental material comprise the subset of genes whose loss resulted in the greatest sensitivity to oxalic acid (MICs of 2 mM). This collection of 105 putative mutants almost certainly contains false positives, but because only half were subsequently subjected to a quantitative assessment of sensitivity to oxalate (cell yield measurements), it is possible that a number are bone fide but unconfirmed mutants.

Confirmed oxalate mutants.
Table 1 lists the 31 genes whose loss resulted in significantly reduced cell yields in YEPD (pH 3) plus 30 mM oxalate relative to those in YEPD (pH 3). The genes are listed alphabetically by ORF within biological process categories (http://db.yeastgenome.org/cgi-bin/GO/goTermMapper). The corresponding deletion mutants were among the 50 or so assayed quantitatively for cell yield because a preliminary measure of growth rates in 96-well plates suggested substantial differences among this group of putative mutants. Genes known to be involved in multiple processes are listed in multiple categories. Mutants previously identified in published screens for sensitivity to sorbic (22, 30) or citric (20) acid are designated with an "S" or "C," respectively. Approximately 35% of these oxalate mutants were not identified in previous screens for sensitivity to 1 or 2 mM sorbic acid, pH 4.5 (22, 30), or to 400 mM citric acid, pH 3.5 (20). Gene functions are indicated if known, as are Arabidopsis thaliana and S. sclerotiorum orthologs and their respective probability scores. The five entries listed in bold are the subset of genes whose loss led to poor survival in YEPD (pH 3) plus 30 mM oxalate relative to that in YEPD (pH 3). The single gene ontology process category that was found to be significantly overrepresented (P = 0.0001) among these confirmed mutants compared to all verified yeast ORFs was "endosome transport." This analysis excluded three of the oxalate mutants, because one of the implicated genes, MTQ2, has not yet been characterized and two others, VPS61 and VPS65, have been designated "dubious." Thus, while 6 of 28 of the verified genes among the oxalate mutants (20%) play roles in endosome transport, only 48 among a total of 4,553 verified yeast ORFs (1%) have been implicated in this process.

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TABLE 1. Yeast genes whose loss results in oxalate sensitivity

Table 2 lists cell yields as A₆₀₀ values for the 31 mutants after 3 days of growth in 500 µl of YEPD (pH 3) (control) or in YEPD (pH 3) plus 30 mM oxalate. Differences in yields in these two media for all mutants were found to be significant (P < 0.05 for Student's t test), whereas differences for the two parent strains, BY4742 and BY4739, were not. Because growth was initiated by diluting overnight cultures grown in control medium about 200-fold into YEPD (pH 3) and into YEPD (pH 3) plus 30 mM oxalate, only about seven to eight doublings were required to attain final cell densities.

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TABLE 2. Cell yields in control medium with or without oxalate

No differences in growth rates observed as function of oxalate.
The growth rates of four chosen mutants, the ada2, snq2, ptc1, and hom6 mutants, and their parent, BY4742, were measured in YEPD (pH 3), YEPD (pH 3) plus 10 mM oxalate, and in YEPD (pH 3) plus 30 mM oxalate. While no differences in growth rates were detected for any of these strains as a function of oxalate (P > 0.05; analysis of variance), differences were observed as a function of genotype. Although both the ptc1 and hom6 mutants grew significantly more slowly than BY4742 in the oxalate-free control medium, this difference was not observed in either the 10 or the 30 mM oxalate medium. And while BY4742 was found to have a doubling time of 2.5 h ± 4% in the oxalate-free control medium, its doubling time increased insignificantly—12%—in YEPD (pH 3) plus either 10 or 30 mM oxalate (P > 0.05, Students' t test; data not shown). The slowest-growing mutant whose doubling time was measured, the ptc1 mutant, was found to have significantly slower growth than BY4742, 3.6 h ± 5% in the control medium, but to have insignificantly faster growth than either BY4742 or itself in YEPD (pH 3) plus 10 or 30 mM oxalate—3.2 h ± 11% (P > 0.05, Students' t test; data not shown).

Oxalate causes cell death.
Cell survival (Table 3) was assessed as a function of 3-day exposure to YEPD (pH 3) plus oxalate, acetate, malonate, propionate, and formate for all strains which exhibited sensitivity in the initial preliminary assay of survival in YEPD (pH 3) plus 30 mM oxalate relative to YEPD (pH 3). Cell densities (cells/ml) of the overnight oxalate-free cultures reached 2 x 10⁸ (BY4742 and BY4739), 10⁸ (vps51 and ric1 mutants), 3 x 10⁷ (vps16 and drs2 mutants), and 6 x 10⁶ (rib4 mutant) prior to a 200-fold dilution into YEPD (pH 3) and into acid-containing media. Assuming all inoculated cells were viable and no subsequent growth occurred, the minimum initial cell count at the start of the 3-day incubation would have been (cells/4 µl) 4 x 10³ (BY4742 and BY4739), 2 x 10³ (vps51 and ric1 mutants), 6 x 10² (vps16 and drs2 mutants), and 10² (rib4 mutant). Thus, the minimum extent of killing that would have resulted in no viable cells surviving in the undiluted 4-µl aliquots plated 3 days later, which was the chosen criterion for designating sensitivity, would have been >99.9% for BY4742 and BY4739 and the vps51 and ric1 mutants and >99% for the vps16, drs2, and rib4 mutants. With respect to oxalate, only the parent strains survived the 3-day incubation with a 30 mM concentration. The vps16 mutant displayed partial sensitivity to 5 mM oxalate, while the rib4 mutant failed to survive the 3-day exposure to even 1 mM oxalate. Figure 1 is a representative YEPD agar plate containing aliquots of cells of the BY4742 parent and ada2 and ric1 mutants incubated in YEPD (pH 3) (control) and in oxalate-containing media for 3 days prior to plating. Only the ric1 mutant exposed to 30 mM oxalate failed to survive. While no strain was found to be sensitive to killing by malonate, all mutants except for the ric1 mutant were found to be sensitive to 30 mM propionate. The vps16 and rib4 mutants exhibited sensitivity to 30 mM acetate, and all strains including the two parents failed to survive exposure to 30 mM formate. With the exception of the rib4 mutant, all the mutants found to be sensitive to oxalate-induced killing by this survival assay are impaired in vesicle-mediated transport, suggesting that this is the key cellular target for oxalate toxicity in yeast.

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TABLE 3. Cell survival following exposure to YEPD (pH 3) supplemented with organic acidsa

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FIG. 1. Survival of cells incubated in presence of oxalate. (A) From left to right, duplicate 4-µl aliquots of undiluted or 10-fold-diluted cells of the indicated strains were spotted on YEPD agar following 3 days of incubation in YEPD (pH 3) (control), YEPD (pH 3) plus 1 mM oxalate, YEPD (pH 3) plus 5 mM oxalate, or YEPD (pH 3) plus 30 mM oxalate. Note the absence of growth of ric1 cells incubated in the presence of 30 mM oxalate (boxed region), while the BY4742 parent and the ada2 mutant grew at all oxalate concentrations tested. (B) Conditions of incubation prior to spotting aliquots of cells are indicated in the corresponding positions for each strain.

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The relative specificity of oxalate-mediated toxicity in yeast with respect to target process is striking, in contrast to the wider range of processes implicated in previous screens for yeast mutants sensitive to sorbic (22, 30) and citric (20) acids. In the case of the oxalate-sensitive mutants, only genes involved in "endosomal transport" were found to be significantly overrepresented relative to those involved in other cellular processes. In support of oxalate interfering specifically with endosomal transport, it is of interest that two mutants identified independently in the present screen, the ric1 and rgp1 mutants, were previously reported to associate in a complex that binds a Golgi-associated GTPase, Ypt6, presumably required for efficient fusion of endosome-derived vesicles with the Golgi apparatus (32). Similarly, two other genes identified independently in the present screen, snf7 and vps20, were previously reported to encode interacting proteins that are members of the ESCRT-III complex (endosomal sorting complex required for transport) (1).

The extreme sensitivity of the rib4 mutant to oxalate suggests interference with riboflavin uptake based on the recent identification of its facilitator, the putative monocarboxylate transporter homolog, Mch5 (28). Previous efforts to identify substrates for Mch5 had ruled out lactate, pyruvate, and acetate (21). We observed that while the rib4 mutant grew reasonably well in YEPD, it reached a noticeably lower cell density in YEPD (pH 3). In YEPD (pH 3) containing 30 mM oxalate, it reached about 4% of this cell density (Table 2). Thus, it is possible that oxalate is an inhibitor of riboflavin uptake by Mch5. Alternatively, oxalate may be a substrate for Mch5. In support of the latter possibility, Mch5 shares significant homology (3 x e^–59) to a putative oxalate/formate antiporter from Aspergillus fumigatus (NCBI accession no. XP_746859). Reihl and Stolz (28) reported that the pH optimum for Mch5 is 7.5, which is consistent with weaker growth of the rib4 mutant at pH 3 than in the less acidic unbuffered YEPD. If this were true, one would predict that all riboflavin mutants ought to be similarly inhibited by oxalate. Upon reviewing the mutants included in the deletion library to determine why no other oxalate-sensitive rib mutants were isolated, we discovered that the rib4 mutant is the only one present, presumably because it is the only riboflavin auxotroph able to grow in YEPD not supplemented with additional riboflavin. Reihl and Stolz (28) show that this is true for the rib5 mutant and state that it is also true for the rib3 and rib7 mutants. Interestingly, Kis et al. (16) reported that the reaction catalyzed by Rib4 can occur nonenzymatically under physiologic conditions, which Reihl and Stolz (28) suggest may explain the less-severe phenotype relative to other riboflavin auxotrophs. This observation would also be consistent with our isolation of the gly1 mutant in the present screen for oxalate-sensitive mutants. GLY1 encodes threonine aldolase, which converts threonine to glycine and acetaldehyde. Glycine is a precursor of purine biosynthesis and hence riboflavin as well. Monschau et al. (23) reported that overexpression of GLY1 combined with threonine supplementation significantly increased riboflavin production in Ashbya gossypii. Thus, the gly1 mutant may behave as a leaky riboflavin auxotroph which in the presence of oxalate would be unable to take up sufficient riboflavin to support growth.

Yeast genes relevant to fungal diseases of plants.
The present study was motivated by an interest in using yeast to discover plant or fungal orthologs relevant to disease interactions between plants and their oxalate-secreting fungal pathogens. Were the results informative? Nineteen S. sclerotiorum orthologs and 18 A. thaliana orthologs were identified by comparing gene sequences of the 31 yeast mutants sensitive to oxalic acid to S. sclerotiorum and A. thaliana genome databases, respectively (Table 1). Four of these orthologs are involved in transport processes, specifically in vesicle trafficking (VPS20, SNF7, VPS16, and DRS2). Significantly, a gene belonging to the DRS2 (9) family of aminophospholipid translocases has been shown to be required for rice blast disease and for induction of host resistance (7). An additional six genes, not including those implicated in transport, function in organelle organization and biogenesis (ADA2, CNM67, KEM1, PTC1, RNR1, and VMA5). Deletion of PTC1, the gene encoding protein phosphatase 2C, was found to increase sensitivity to oxalate. Mollapour et al. (22) reported that the deletion caused sensitivity to sorbate. Ptc1 inactivates the high-osmolarity glycerol pathway by dephosphorylating the mitogen-activated protein kinase Hog1 (37). The high-osmolarity glycerol pathway has previously been suggested to be involved in adaptation to citric acid stress (20). Moreover, p38, the mammalian homolog of Hog1, has been implicated in signal transduction of oxalate stress in renal epithelial cells (12, 18). Specific protein phosphatase 2C genes (ABI1 and ABI2) in A. thaliana control ABA signaling and transpiration (24), and Guimaraes and Stotz (11) have shown that the abi1 mutation increases susceptibility to an oxalate-deficient mutant of S. sclerotiorum.

Loss of SNQ2 resulted in sensitivity to oxalate but was not previously identified in screens for citrate or sorbate sensitivity. SNQ2 encodes an ABC transporter closely related to the Arabidopsis gene PEN3/PDR8, which was recently shown to contribute to nonhost resistance to penetration by fungal pathogens (35). It is conceivable that SNQ2 exports oxalic acid or a toxic metabolite that accumulates intracellularly as a result of oxalate poisoning. Multicopy SNQ2 is known to confer resistance to N-nitroquinoline-N-oxide and other xenobiotics (31). The A. thaliana ortholog of yeast RIB4 is COS1, which has been shown to be involved in jasmonate signaling and was identified as a suppressor of COI1, an F-box protein and essential regulator of jasmonate responses (39). This suggests a possible relationship between oxalate tolerance in yeast and jasmonate-related defense signaling in plants.

The present study has identified endosomal transport as the major target of oxalate-induced toxicity in yeast. Through a physiologic quirk of rib4 auxotrophy, the study has also provided indirect evidence that oxalate is either an inhibitor or a substrate of the putative monocarboxylate transporter, Mch5. The study has also identified candidate plant genes previously associated with plant host defense but not previously associated with oxalic acid tolerance or a specific response to oxalic acid-secreting fungal pathogens. This is significant because genetic analysis of oxalate sensitivity in plants has been experimentally difficult (H. Stotz, unpublished data), in part due to the quantitative inheritance and low heritability of this trait (17).

 
We thank Hyatt Green and Vihangi Hindagolla for technical assistance, Hagai Abeliovich and James Osborne for helpful discussions, and Hagai Abeliovich and Gary Merrill for reviewing an earlier version of the manuscript. The OSU Environmental Health Sciences Center, Oregon State University (grant number P30 ES00210; NIEHS, NIH), is thanked for providing the yeast deletion library. The Broad Institute is gratefully acknowledged for early access to the S. sclerotiorum genome database.

The USDA-ARS National Sclerotinia Initiative provided partial financial support.

 
* Corresponding author. Mailing address: Department of Food Science and Technology, Wiegand Hall, Oregon State University, Corvallis, OR 97331-6602. Phone: (541) 737-6510. Fax: (541) 737-1877. E-mail: alan.bakalinsky{at}oregonstate.edu

Published ahead of print on 20 July 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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1
1. Babst, M., D. J. Katzmann, E. J. Estepa-Sabal, T. Meerloo, and S. D. Emr. 2002. ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting. Dev. Cell 3:271-282.[CrossRef][Medline]
2
2. Baldwin, H. 1900. An experimental study of oxaluria, with special reference to its fermentative origin. J. Exp. Med. 5:27-46.[Abstract/Free Full Text]
3
3. Bateman, D. F., and S. V. Beer. 1965. Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis of Sclerotium rolfsii. Phytopathology 55:204-211.[Medline]
4
4. Cessna, S. G., V. E. Sears, M. B. Dickman, and P. S. Low. 2000. Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191-2199.[Abstract/Free Full Text]
5
5. Dutton, M. V., and C. S. Evans. 1996. Oxalate production by fungi: its role in pathogenicity and ecology in the soil environment. Can. J. Microbiol. 42:881-895.[CrossRef]
6
6. Dutton, M. V., C. S. Evans, P. T. Atkey, and D. A. Wood. 1993. Oxalate production by Basidiomycetes, including the white-rot species Coriolus versicolor and Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 39:5-10.
7
7. Gilbert, M. J., C. R. Thornton, G. E. Wakley, and N. J. Talbot. 2006. A P-type ATPase required for rice blast disease and induction of host resistance. Nature 440:535-539.[CrossRef][Medline]
8
8. Godoy, G., J. R. Steadman, M. B. Dickman, and R. Dam. 1990. Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiol. Mol. Plant Pathol. 37:179-191.[CrossRef]
9
9. Graham, T. R. 2004. Flippases and vesicle-mediated protein transport. Trends Cell Biol. 14:670-677.[CrossRef][Medline]
10
10. Graustein, W. C., K. J. Cromack, and P. Sollins. 1977. Calcium oxalate: occurrence in soils and effect on nutrient and geochemical cycles. Science 198:1252-1254.[Abstract/Free Full Text]
11
11. Guimaraes, R. L., and H. U. Stotz. 2004. Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection. Plant Physiol. 136:3703-3711.[Abstract/Free Full Text]
12
12. Han, H. J., M. J. Lim, and Y. J. Lee. 2004. Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and Cpla2 signaling pathways. Am. J. Physiol. Cell Physiol. 287:C1058-C1066.[Abstract/Free Full Text]
13
13. Holmes, R. P., and D. G. Assimos. 1998. Glyoxylate synthesis, and its modulation and influence on oxalate synthesis. J. Urol. 160:1617-1624.[CrossRef][Medline]
14
14. Hu, X., D. L. Bidney, N. Yalpani, J. P. Duvick, O. Crasta, O. Folkerts, and G. Lu. 2003. Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiol. 133:170-181.[Abstract/Free Full Text]
15
15. Kesarwani, M., M. Azam, K. Natarajan, A. Mehta, and A. Datta. 2000. Oxalate decarboxylase from Collybia velutipes: molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. J. Biol. Chem. 275:7230-7238.[Abstract/Free Full Text]
16
16. Kis, K., K. Kugelbrey, and A. Bacher. 2001. Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions. J. Org. Chem. 66:2555-2559.[CrossRef][Medline]
17
17. Kolkman, J. M., and J. D. Kelly. 2003. QTL conferring resistance and avoidance to white mold in common bean. Crop Sci. 43:539-548.[Abstract/Free Full Text]
18
18. Koul, H. K., M. Menon, L. S. Chaturvedi, S. Koul, A. Sekhon, A. Bhandari, and M. Huang. 2002. COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells. J. Biol. Chem. 277:36845-36852.[Abstract/Free Full Text]
19
19. Lane, B. G., J. M. Dunwell, J. A. Ray, M. R. Schmitt, and A. C. Cuming. 1993. Germin, a protein marker of early plant development, is an oxalate oxidase. J. Biol. Chem. 268:12239-12242.[Abstract/Free Full Text]
20
20. Lawrence, C. L., C. H. Botting, R. Atrobus, and P. J. Coote. 2004. Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress. Mol. Cell. Biol. 24:3307-3323.[Abstract/Free Full Text]
21
21. Makuc, J., S. Paiva, M. Schauen, R. Kramer, B. Andre, M. Casal, C. Leao, and E. Boles. 2001. The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane. Yeast 18:1131-1143.[CrossRef][Medline]
22
22. Mollapour, M., D. Fong, K. Balakrishnan, N. Harris, S. Thompson, C. Schuller, K. Kuchler, and P. W. Piper. 2004. Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative. Yeast 21:927-946.[CrossRef][Medline]
23
23. Monschau, N., H. Sahm, and K.-P. Stahmann. 1998. Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii. Appl. Environ. Microbiol. 64:4283-4290.[Abstract/Free Full Text]
24
24. Murata, Y., Z.-M. Pei, I. C. Mori, and J. Schroeder. 2001. Abscisic acid activation of plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. Plant Cell 13:2513-2523.[Abstract/Free Full Text]
25
25. Nakagawa, Y., K. Shimazu, M. Ebihara, and K. Nakagawa. 1999. Aspergillus niger pneumonia with fatal pulmonary oxalosis. J. Infect. Chemother. 5:97-100.[CrossRef][Medline]
26
26. Nakata, P. A. 2003. Advances in our understanding of calcium oxalate crystal formation and function in plants. Plant Sci. 164:901-909.
27
27. Noyes, R. D., and J. G. Hancock. 1981. Role of oxalic acid in the Sclerotinia sclerotiorum wilt of sunflower Helianthus annuus. Physiol. Plant Pathol. 18:123-132.
28
28. Reihl, P., and J. Stolz. 2005. The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B₂) uptake in Saccharomyces cerevisiae. J. Biol. Chem. 280:39809-39817.[Abstract/Free Full Text]
29
29. Schmidt, C. J., B. K. Whitten, and D. D. Nicholas. 1981. A proposed role for oxalic acid in non-enzymatic wood decay by brown-rot fungi. Proc. Annu. Meet. Am. Wood Preserv. Assoc. 77:157-164.
30
30. Schüller, C., Y. M. Mamnun, M. Mollapour, G. Krapf, M. Schuster, B. E. Bauer, P. W. Piper, and K. Kuchler. 2004. Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae. Mol. Biol. Cell 15:706-720.[Abstract/Free Full Text]
31
31. Servos, J., E. Haase, and M. Brendel. 1993. Gene SNQ2 of Saccharomyces cerevisiae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169 kDa protein homologous to ATP-dependent permeases. Mol. Gen. Genet. 238:214-218.
32
32. Siniossoglou, S., S. Y. Peak-Chew, and H. R. B. Pelham. 2000. Ric1p and Rgp1p form a complex that catalyzes nucleotide exchange on Ypt6p. EMBO J. 19:4885-4894.[CrossRef][Medline]
33
33. Sniegowski, P. D., P. G. Dombrowski, and E. Fingerman. 2002. Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics. FEMS Yeast Res. 1:299-306.[Medline]
34
34. Sperry, J. S., and M. T. Tyree. 1988. Mechanism of water stress-induced xylem embolism. Plant Physiol. 88:581-587.[Abstract/Free Full Text]
35
35. Stein, M., J. Dittgen, C. Sanchez-Rodriguez, B.-H. Hou, A. Molina, P. Schulze-Lefert, V. Lipka, and S. Somerville. 2006. Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration. Plant Cell 18:731-746.[Abstract/Free Full Text]
36
36. Sutter, H.-P., and G. E. B. Jones. 1985. Interactions between copper and wood degrading fungi, p. 29-41. Record of the Annual Convention of the British Wood Preserving Association. British Wood Preserving Association, London, United Kingdom.
37
37. Warmka, J., J. Hanneman, J. Lee, D. Amin, and I. Ota. 2001. Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1. Mol. Cell. Biol. 21:51-60.[Abstract/Free Full Text]
38
38. Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K. Anderson, B. Andre, R. Bangham, R. Benito, J. D. Boeke, H. Bussey, A. M. Chu, C. Connelly, K. Davis, F. Dietrich, S. W. Dow, M. El Bakkoury, F. Foury, S. H. Friend, E. Gentalen, G. Giaever, J. H. Hegemann, T. Jones, M. Laub, H. Liao, N. Liebundguth, D. J. Lockhart, A. Lucau-Danila, M. Lussier, N. M'Rabet, P. Menard, M. Mittmann, C. Pai, C. Rebischung, J. L. Revuelta, L. Riles, C. J. Roberts, P. Ross-MacDonald, B. Scherens, M. Snyder, S. Sookhai-Mahadeo, R. K. Storms, S. Véronneau, M. Voet, G. Volckaert, T. R. Ward, R. Wysocki, G. S. Yen, K. Yu, K. Zimmermann, P. Philippsen, M. Johnston, and R. W. Davis. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901-906.[Abstract/Free Full Text]
39
39. Xiao, S., L. Dai, F. Liu, Z. Wang, W. Peng, and D. Xie. 2004. COS1: An Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence. Plant Cell 16:1132-1142.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, September 2007, p. 5919-5927, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.02843-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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