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

Abstract

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 rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.

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.

MATERIALS AND METHODS

Yeast strains and media.

The S. cerevisiae deletion library YSC1054Y, constructed with parent strain BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 or BY4739 MATα leu2Δ0 lys2Δ0 ura3Δ0, 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 ada2Δ, hom6Δ, ptc1Δ, and snq2Δ 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.

RESULTS

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.

TABLE 1.

Yeast genes whose loss results in oxalate sensitivity

Genea	Yeast ORF	S/Cb	Function	Arabidopsis ortholog		Sclerotinia ortholog
				Accession no.c	E-valued	Accession no.c	E-valued
Organelle organization and biogenesis
ADA2	YDR448W	n/n	Chromatin modification, histone acetylation	At4g16420	3.00E−54	SS1G14454.1	3.00E−82
CNM67	YNL225C	y/n	Microtubule nucleation	At3g19370	6.00E−08	SS1G03275.1	4.00E−08
ERG2	YMR202W	y/y	C-8 sterol isomerase			SS1G06738.1	5.00E−43
GON7	YJL184W	y/n	Cell wall mannoprotein biosynthesis				No hits found
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
PTC1	YDL006W	y/n	Hog pathway, protein phosphatase	At5g24940	2.00E−33	SS1G00528.1	4.00E−48
RNR1	YER070W	y/n	Ribonucleotide-diphosphate reductase, large subunit	At2g21790	0.0	SS1G10937.1	0.0
SNF7	YLR025W	y/n	Endosomal sorting complex subunit	At2g19830	2.00E−09	SS1G10606.1	6.00E−19
VAM10	YOR068C	n/n	Vacuole fusion, nonautophagic				No hits found
VMA5	YKL080W	y/y	Subunit C of vacuolar H+-ATPase	At1g12840	9.00E−49	SS1G14389.1	6.00E−47
VMA22	YHR060W	y/y	Required for vacuolar H+(V-ATPase) function				No hits found
VPS16	YPL045W	y/y	Subunit of vacuole fusion and vacuole protein sorting complex	At2g38020	4.00E−42	SS1G00520.1	2.00E−36
VPS51	YKR020W	n/n	Golgi apparatus-associated retrograde protein complex component				No hits found
Transport
DRS2	YAL026C	n/y	Vesicle trafficking	At1g59820	0.0	SS1G08813.1	0.0
RCS1	YGL071W	n/y	Transcription factor involved in Fe homeostasis				No hits found
RGP1	YDR137W	n/n	Subunit of Golgi membrane exchange factor (Ric1p-Rgp1p)				No hits found
RIC1	YLR039C	n/n	Involved in retrograde transport to cis-Golgi network				No hits found
SNF7	YLR025W	y/n	Endosomal sorting complex subunit	At2g19830	2.00E−09	SS1G10606.1	6.00E−19
VPS16	YPL045W	y/y	Subunit of vacuole fusion and vacuole protein sorting complex	At2g38020	4.00E−42	SS1G00520.1	2.00E−36
VPS20	YMR077C	n/n	Myristoylated subunit of endosomal sorting complex	At5g63880	2.00E−15		No hits found
VPS51	YKR020W	n/n	Golgi apparatus-associated retrograde protein complex component				No hits found
VPS61	YDR136C	n/n	Vacuolar protein sorting				No hits found
VPS65	YLR322W	y/n	Vacuolar protein sorting				No hits found
RNA metabolic process
CCR4	YAL021C	n/n	Component of CCR4-NOT transcriptional regulatory complex	At3g58560	5.00E−46	SS1G12598.1	4.00E−94
CDC40	YDR364C	y/n	Pre-mRNA splicing factor	At1g10580	7.00E−72	SS1G13123.1	7.00E−78
CTK3	YML112W	y/n	Kinase subunit that phosphorylates large subunit of RNA polymerase II				No hits found
DRS2	YAL026C	n/y	Vesicle trafficking	At1g59820	0.0	SS1G08813.1	0.0
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
POP2	YNR052C	y/n	RNase of the DEDD superfamily	At2g32070	2.00E−49	SS1G12459.1	3.00E−36
PTC1	YDL006W	y/n	Hog pathway	At5g24940	2.00E−33	SS1G00528.1	4.00E−48
RCS1	YGL071W	n/y	Transcription factor involved in Fe homeostasis				No hits found
Vesicle-mediated transport
DRS2	YAL026C	n/y	Vesicle trafficking	At1g59820	0.0	SS1G08813.1	0.0
RIC1	YLR039C	n/n	Involved in retrograde transport to cis-Golgi network				No hits found
RGP1	YDR137W	n/n	Subunit of Golgi membrane exchange factor (Ric1p-Rgp1p)				No hits found
SNF7	YLR025W	y/n	Endosomal sorting complex subunit	At2g19830	2.00E−09	SS1G10606.1	6.00E−19
VPS16	YPL045W	y/y	Subunit of vacuole fusion and vacuole protein sorting complex	At2g38020	4.00E−42	SS1G00520.1	2.00E−36
VPS20	YMR077C	n/n	Myristoylated subunit of endosomal sorting complex	At5g63880	2.00E−15		No hits found
VPS51	YKR020W	n/n	Golgi apparatus-associated retrograde protein complex component				No hits found
Transcription
CCR4	YAL021C	n/n	Component of CCR4-NOT transcriptional complex	At3g58560	5.00E−46	SS1G12598.1	4.00E−94
CTK3	YML112W	y/n	Kinase subunit that phosphorylates large subunit of RNA polymerase II				No hits found
GON7	YJL184W	y/n	Cell wall mannoprotein biosynthesis				No hits found
POP2	YNR052C	y/n	RNase of the DEDD superfamily	At2g32070	2.00E−49	SS1G12459.1	3.00E−36
RCS1	YGL071W	n/y	Transcription factor involved in Fe homeostasis				No hits found
Cell cycle
CCR4	YAL021C	n/n	Component of CCR4-NOT transcriptional regulatory complex	At3g58560	5.00E−46	SS1G12598.1	4.00E−94
CDC40	YDR364C	y/n	Pre-mRNA splicing factor	At1g10580	7.00E−72	SS1G13123.1	7.00E−78
CNM67	YNL225C	y/n	Microtubule nucleation	At3g19370	6.00E−08	SS1G03275.1	4.00E−08
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
Protein modification process
ADA2	YDR448W	n/n	Chromatin modification, histone acetylation	At4g16420	3.00E−54	SS1G14454.1	3.00E−82
CTK3	YML112W	y/n	Kinase subunit that phosphorylates large subunit of RNA polymerase II				No hits found
MTQ2	YDR140W	n/n	Peptidyl-glutamine methylation (eRF1)	At3g13440	8.00E−15	SS1G14261.1	9.00E−33
PTC1	YDL006W	y/n	Hog pathway, protein phosphatase	At5g24940	2.00E−33	SS1G00528.1	4.00E−48
Membrane organization and biogenesis
DRS2	YAL026C	n/y	Vesicle trafficking	At1g59820	0.0	SS1G08813.1	0.0
VAM10	YOR068C	n/n	Vacuole fusion, non-autophagic				No hits found
VPS16	YPL045W	y/y	Subunit of vacuole fusion and vacuole protein sorting complex	At2g38020	4.00E−42	SS1G00520.1	2.00E−36
Response to stress
GON7	YJL184W	y/n	Cell wall mannoprotein biosynthesis				No hits found
PTC1	YDL006W	y/n	Hog pathway, protein phosphatase	At5g24940	2.00E−33	SS1G00528.1	4.00E−48
SNQ2	YDR011W	n/n	ABC transporter conferring multidrug and singlet O resistance	At1g15210	1.00E−110	SS1G02042.1	0.0
Lipid metabolism
DRS2	YAL026C	n/y	Vesicle trafficking	At1g59820	0.0	SS1G08813.1	0.0
ERG2	YMR202W	y/y	C-8 sterol isomerase			SS1G06738.1	5.00E−43
ERG24	YNL280c		C-14 sterol reductase			SS1G07099.1	0.00E+00
Amino acid and derivative metabolic process
GLY1	YEL046C	y/y	Threonine aldolase	At3g04520	6.00E−45	SS1G01438.1	6.00E−70
HOM6	YJR139C	n/n	Homoserine dehydrogenase	At1g31230	3.00E−58	SS1G14424.1	2.00E−86
DNA metabolic process
ADA2	YDR448W	n/n	Chromatin modification, histone acetylation	At4g16420	3.00E−54	SS1G14454.1	3.00E−82
RNR1	YER070W	y/n	Ribonucleotide-diphosphate reductase, large subunit	At2g21790	0.0	SS1G10937.1	0.0
Protein catabolic process
SNF7	YLR025W	y/n	Endosomal sorting complex subunit	At2g19830	2.00E−09	SS1G10606.1	6.00E−19
VPS20	YMR077C	n/n	Myristoylated subunit of endosomal sorting complex	At5g63880	2.00E−15		No hits found
Cell homeostasis
VMA5	YKL080W	y/y	Subunit C of vacuolar H+-ATPase	At1g12840	9.00E−49	SS1G14389.1	6.00E−47
VMA22	YHR060W	y/y	Required for vacuolar H+(V-ATPase) function				No hits found
Generation of precursor metabolites and energy
VMA22	YHR060W	y/y	Required for vacuolar H+(V-ATPase) function				No hits found
Cell wall organization and biogenesis
GON7	YJL184W	y/n	Cell wall mannoprotein biosynthesis				No hits found
Nuclear organization and biogenesis
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
Signal transduction
PTC1	YDL006W	y/n	Hog pathway, protein phosphatase	At5g24940	2.00E−33	SS1G00528.1	4.00E−48
Cytoskeleton organization and biogenesis
CNM67	YNL225C	y/n	Microtubule nucleation	At3g19370	6.00E−08	SS1G03275.1	4.00E−08
Vitamin metabolic process
RIB4	YOL143Ce	y/n	Lumazine synthase, precursor of riboflavin	At2g44050	2.00E−14	SS1G04679.1	3.00E−25
Cell budding
VPS51	YKR020W	n/n	Golgi apparatus-associated retrograde protein complex component				No hits found
Ribosome biogenesis and assembly
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
Conjugation
KEM1	YGL173C	y/n	Exonuclease component of cytoplasmic processing bodies	At1g75660	1.00E−122	SS1G07676.1	0.0
Carbohydrate metabolic process
VMA22	YHR060W	y/y	Required for vacuolar H+(V-ATPase) function				No hits found
Anatomical structure morphogenesis
VPS51	YKR020W	n/n	Golgi apparatus-associated retrograde protein complex component				No hits found
Other
VMA21	YGR105W	y/n	Required for vacuolar H+(V-ATPase) function				No hits found

^aEntries in bold font are the subset of 5 genes whose loss resulted in poor survival after 3 days in YEPD (pH 3) plus 30 mM oxalate relative to that in YEPD (pH 3). Genes are listed alphabetically within biological process categories (http://db.yeastgenome.org/cgi-bin/GO/goTermMapper) and may appear in more than one.

^bOxalate mutants isolated (y) or not (n) in previous screens for sorbate-sensitive mutants (2 mM, pH 4.5 [22]; 1 mM, pH 4.5 [30]) and citrate-sensitive mutants (400 mM, pH 3.5 [20]).

^cLocus in Arabidopsis and Sclerotinia.

^dE-value for Arabidopsis and Sclerotinia orthologs.

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.

TABLE 2.

Cell yields in control medium with or without oxalate

Strain name or mutated gene	Mean A600 value (SD) for cell yield ina:		P value (t test)
	Control	Oxalate (30 mM)
BY4742	4.87 (0.36)	4.87 (0.26)	1.000
BY4739	5.08 (0.01)	4.23 (0.76)	0.356
ada2	4.37 (0.08)	4.09 (0.01)	0.039
aft1	4.57 (0.04)	2.41 (0.01)	<0.001
ccr4	2.83 (0.01)	0.07 (0.00)	0.002
cdc40	1.22 (0.08)	0.08 (0.01)	0.003
cnm67	3.41 (0.04)	1.60 (0.27)	0.011
ctk3	2.40 (0.06)	0.06 (0.00)	0.011
drs2	2.59 (0.16)	0.05 (0.01)	0.002
erg2	4.71 (0.21)	3.92 (0.06)	0.035
erg24	4.40 (0.31)	0.12 (0.03)	0.003
gly1	3.90 (0.00)	3.52 (0.04)	0.006
gon7	2.77 (0.21)	0.03 (0.01)	0.033
hom6	4.02 (0.30)	2.80 (0.13)	0.033
kem1	2.32 (0.06)	0.07 (0.01)	<0.001
mtq2	4.21 (0.05)	0.14 (0.05)	<0.001
pop2	2.17 (0.20)	0.07 (0.01)	0.042
ptc1	3.63 (0.01)	3.18 (0.00)	<0.001
rgp1	4.34 (0.01)	0.15 (0.09)	<0.001
rib4	3.06 (0.28)	0.13 (0.04)	0.005
ric1	4.63 (0.39)	0.08 (0.01)	0.038
rnr1	3.36 (0.00)	0.10 (0.02)	0.003
snf7	3.72 (0.20)	0.08 (0.01)	0.001
snq2	5.45 (0.01)	4.84 (0.16)	0.034
vam10	3.79 (0.20)	0.18 (0.08)	0.002
vma21	3.32 (0.22)	1.10 (0.06)	0.005
vma22	2.24 (0.06)	0.16 (0.08)	0.001
vma5	4.12 (0.25)	0.18 (0.14)	0.003
vps16	2.32 (0.08)	0.09 (0.01)	0.001
vps20	4.07 (0.30)	0.19 (0.17)	0.004
vps51	4.62 (0.150	0.11 (0.01)	0.014
vps61	4.64 (0.08)	0.15 (0.08)	<0.001
vps65	3.24 (0.23)	0.29 (0.01)	0.003

^aValues are A₆₀₀ measurements of 10-fold-diluted duplicate cultures multiplied by 10. Control, YEPD (pH 3); oxalate, YEPD (pH 3) plus 30 mM oxalate.

No differences in growth rates observed as function of oxalate.

The growth rates of four chosen mutants, the ada2Δ, snq2Δ, ptc1Δ, and hom6Δ 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 ptc1Δ and hom6Δ 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 ptc1Δ 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 × 10⁸ (BY4742 and BY4739), 10⁸ (vps51Δ and ric1Δ mutants), 3 × 10⁷ (vps16Δ and drs2Δ mutants), and 6 × 10⁶ (rib4Δ 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 × 10³ (BY4742 and BY4739), 2 × 10³ (vps51Δ and ric1Δ mutants), 6 × 10² (vps16Δ and drs2Δ mutants), and 10² (rib4Δ 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 vps51Δ and ric1Δ mutants and >99% for the vps16Δ, drs2Δ, and rib4Δ mutants. With respect to oxalate, only the parent strains survived the 3-day incubation with a 30 mM concentration. The vps16Δ mutant displayed partial sensitivity to 5 mM oxalate, while the rib4Δ 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 ada2Δ and ric1Δ mutants incubated in YEPD (pH 3) (control) and in oxalate-containing media for 3 days prior to plating. Only the ric1Δ 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 ric1Δ mutant were found to be sensitive to 30 mM propionate. The vps16Δ and rib4Δ 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 rib4Δ 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.

TABLE 3.

Cell survival following exposure to YEPD (pH 3) supplemented with organic acids^a

Strain name or genotype	Con	Cell growth on control alone or with acid at indicated concn (mM)
		Oxa			Ace			Mal			Pro			For
		1	5	30	1	5	30	1	5	30	1	5	30	1	5	30
BY4742	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−
BY4739	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	−
drs2Δ	+	+	+	−	+	+	+	+	+	+	+	+	−	+	+	−
vps16Δ	+	+	±	−	+	+	−	+	+	+	+	+	−	+	+	−
vps51Δ	+	+	+	−	+	+	+	+	+	+	+	+	−	+	+	−
ric1Δ	+	+	+	−	+	+	+	+	+	+	+	+	+	+	+	−
rib4Δ	+	−	−	−	+	+	−	+	+	+	+	+	−	+	+	−

^aCon, control medium, YEPD (pH 3.0). Acid-supplemented media were control medium supplemented with 1, 5, or 30 mM of the indicated acid, adjusted to pH 3.0. Oxa, oxalic acid; Ace, acetic acid; Mal, malonic acid; Pro, propionic acid; For, formic acid; +, confluent growth on YEPD agar for both the undiluted and diluted aliquots of cells exposed for 3 days as described in Materials and Methods; ±, weak growth; −, complete absence of growth.

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 ric1Δ cells incubated in the presence of 30 mM oxalate (boxed region), while the BY4742 parent and the ada2Δ 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.

DISCUSSION

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 ric1Δ 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, snf7Δ and vps20Δ, 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 rib4Δ 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 rib4Δ 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 × 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 rib4Δ 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 rib4Δ 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 rib5Δ mutant and state that it is also true for the rib3Δ and rib7Δ 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 gly1Δ 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 gly1Δ 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 rib4Δ 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).