Aberrant synthesis of indole-3-acetic acid in Saccharomyces cerevisiae triggers morphogenic transition, a virulence trait of pathogenic fungi.
ABSTRACT Many plant-associated microbes synthesize the auxin indole-3-acetic acid (IAA), and several IAA biosynthetic pathways have been identified in microbes and plants. Saccharomyces cerevisiae has previously been shown to respond to IAA by inducing pseudohyphal growth. We observed that IAA also induced hyphal growth in the human pathogen Candida albicans and thus may function as a secondary metabolite signal that regulates virulence traits such as hyphal transition in pathogenic fungi. Aldehyde dehydrogenase (Ald) is required for IAA synthesis from a tryptophan (Trp) precursor in Ustilago maydis. Mutant S. cerevisiae with deletions in two ALD genes are unable to convert radiolabeled Trp to IAA, yet produce IAA in the absence of exogenous Trp and at levels higher than wild type. These data suggest that yeast may have multiple pathways for IAA synthesis, one of which is not dependent on Trp.
- SourceAvailable from: Wei-Ta Fang[Show abstract] [Hide abstract]
ABSTRACT: Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3- acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% Ltryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.PLoS ONE 12/2014; 9(12):e114196. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Pathogenic fungi cause superficial infections but pose a significant public health risk when infections spread to deeper tissues, such as the lung. Within the last three decades, fungi have been identified as the leading cause of nosocomial infections making them the focus of research. This review outlines the model systems such as the mouse, zebrafish larvae, flies, and nematodes, as well as ex vivo and in vitro systems available to study common fungal pathogens.Pathogens (Basel, Switzerland). 01/2014; 3(3):549-562.
- [Show abstract] [Hide abstract]
ABSTRACT: Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.PLoS ONE 12/2014; 9(12):e114196. · 3.53 Impact Factor
Copyright ? 2010 by the Genetics Society of America
Aberrant Synthesis of Indole-3-Acetic Acid in Saccharomyces cerevisiae
Triggers Morphogenic Transition, a Virulence
Trait of Pathogenic Fungi
Reeta Prusty Rao,*,1Ally Hunter,* Olga Kashpur*
and Jennifer Normanly†
*Department of Biology and Biotechnology, Life Sciences and Bioengineering Center at Gateway Park,
Worcester Polytechnic Institute, Worcester, Massachusetts 01605 and†Department of Biochemistry
and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003
Manuscript received December 7, 2009
Accepted for publication February 24, 2010
Many plant-associated microbes synthesize the auxin indole-3-acetic acid (IAA), and several IAA
biosynthetic pathways have been identified in microbes and plants. Saccharomyces cerevisiae has previously
been shown to respond to IAA by inducing pseudohyphal growth. We observed that IAA also induced
hyphal growth in the human pathogen Candida albicans and thus may function as a secondary metabolite
signal that regulates virulence traits such as hyphal transition in pathogenic fungi. Aldehyde
dehydrogenase (Ald) is required for IAA synthesis from a tryptophan (Trp) precursor in Ustilago maydis.
Mutant S. cerevisiae with deletions in two ALD genes are unable to convert radiolabeled Trp to IAA, yet
produce IAA in the absence of exogenous Trp and at levels higher than wild type. These data suggest that
yeast may have multiple pathways for IAA synthesis, one of which is not dependent on Trp.
differentiation (Halliday et al. 2009; Moller and
Weijers 2009; Sundberg and Ostergaard 2009;
Zazimalova et al. 2009; Abel and Athanosios 2010;
McSteen 2010; Scarpella et al. 2010); however, IAA
has been identified in numerous plant-associated
bacteria (reviewed in Glick et al. 1999a,b) and several
fungi, including Rhizopus suinous (Thimann 1935),
Rhizoctonia (Furukawa et al. 1996), Colletotrichum
(Robinson et al. 1998), and yeast (Nielsen 1931;
Gruen 1959). Microbial IAA plays a significant role in
plant–microbe interactions (Glick et al. 1999a), both
pathogenic and symbiotic (Hirsch et al. 1989; Reineke
et al. 2008). Plants infected with pathogenic microbes
manifest phenotypes consistent with elevated levels of
IAA, such as gall formation (a tumor resulting from
cellular proliferation) and lengthening of the stem
(Viglierchio 1971; Barash and Manulis-Sasson
2009; Stewart and Nemhauser 2009). The interplay
between microbial-derived IAA and plant-derived IAA
in plant disease is just beginning to be defined.
Exogenous IAA regulates filamentation in Saccharo-
myces cerevisiae, a fungus that is primarily associated with
plants, by inducing expression of genes that mediate its
morphological transition from a vegetative form to a
HE auxin indole-3-acetic acid (IAA) is best known
for its role in plant cell elongation, division, and
The fungal transcription factor, Yap1, regulates IAA
homeostasis in S. cerevisiae (Prusty et al. 2004) by
downregulating auxin permeases (Avt proteins) that
import IAA in S. cerevisiae (Prusty et al. 2004). We show
here that IAA stimulates filamentation in the human
pathogen Candida albicans and that C. albicans Yap1
(Cap1) also mediates IAA phenotypes. Filamentation
often underlies the development of virulence of C.
albicans. For example, the C. albicans double mutant
cph1D/D efg1D/D is defective in the MAP kinase pathway
through Cph1, as well as in the PKA pathway via Efg1.
This mutant fails to switch from vegetative to filamen-
1999; Liu 2001; Sohn et al. 2003) and is also avirulent
(Dieterich et al. 2002). These studies suggest that the
secondary metabolite IAA is a chemical signal that
regulates fungal pathogenesis.
Plants have multiple pathways to synthesize, inacti-
vate, and catabolize IAA (Delker et al. 2008; Lau et al.
2008; Normanly 2009). Molecular genetic studies in
model systems such as Arabidopsis thaliana (reviewed in
Normanly 2009), coupled with precise analytical meth-
ods (Barkawi et al. 2008), have helped expose some
redundancy within this network. In fungi, IAA has been
generally proposed as a metabolite of tryptophan (Trp)
(Hazelwood et al. 2008) but this has been conclusively
demonstrated only in Ustilago maydis (Reneke et al.
1988) and S. uvarum (Shin et al. 1991). Early studies
used activity assays or qualitative colorimetric techni-
ques to indicate the presence of IAA. Thin layer
Worcester, MA 01609. E-mail: email@example.com
Genetics 185: 211–220 (May 2010)
chromatography (TLC) and high performance liquid
chromatography (HPLC) were subsequently employed
for thedetection ofIAA,where thebioactivecompound
was shown to chromatograph with authentic IAA. De-
finitive isotope dilution quantification of IAA was first
carried out with [14C]IAA and extracts from U. zeae
tumors (Turian and Hamilton 1960).
Here, we used gas-chromatography mass spectrome-
try (GC-MS) coupled with stable isotope dilution to
demonstrate that S. cerevisiae synthesizes IAA. We iden-
tified genes homologous to the aldehyde dehydroge-
pathway in U. maydis (Figure 1) (Basse et al. 1996;
Reineke et al. 2008). Our results are consistent with the
presence of a Trp-independent IAA biosynthetic path-
way in yeast as well.
MATERIALS AND METHODS
Strains, media, and growth conditions: Table 1 lists the
strains used in this study. Deletion strains were derived from
the yeast-deletion set (Winzeler et al. 1999) and subsequently
reconstructed by replacement of the relevant ORF with a
and phenotypic studies were performed in cognate deletion
mutants, made in the S1278b background. A [14C]Trp in-
corporation assay was performed to verify that phenotype
observed in the library strain could be recapitulated in the
newly constructed S1278b strain. Typically three indepen-
dent transformants were isolated, confirmed by PCR, and used
for further studies. Standard culture conditions were used
(Sherman et al. 1986) and analysis of IAA-associated pheno-
types was performed as described earlier (Prusty et al. 2004).
[14C]Trp incorporation assay: Yeast strains were grown in
5-ml overnight cultures with aeration at 30? in synthetic
complete medium (Sigma, St. Louis) (Guthrie and Fink
1991). To estimate cell density, the absorbance at 600 nm was
107cfu/ml). Cells (1 ml) were harvested by centrifugation at
3000 rpm for 5 min on an Eppendorf table-top microfuge at
room temperature. Cells were washed twice by resuspending
pellets in water and then harvested by centrifugation. Cell
pellets were resuspended in 200 ml of SD medium supple-
mented with auxotrophic amino acids (Guthrie and Fink
1991). Samples were incubated with rocking (Thermolyne,
speci mix) at 30? for ?18 hr in media containing 400 mm Trp
and 0.5 mCi of [14C]Trp (Trp L-[side chain-3-14C], specific
activity 50 mCi/mmol; American Radiochemicals). Cells were
removed by centrifugation (3000 rpm in an Eppendorf table-
top microfuge) at room temperature and the conditioned
medium (CM) was transferred to new tubes for TLC. Control
samples were prepared identically but without the addition of
cells to the SD medium. Ten microliters of the CM was spotted
on TLC plates. The [14C]Trp metabolites in the CM were
resolved on a silica gel 60 F254(20 3 20 cm, 250 mm thick,
precoated) TLC plate (EMD Chemicals). A mixture of 85%
chloroform, 14% methanol, and 1% water was used as the
was visualized by autoradiography. Commercially available
[14C]IAA (American Radiochemicals) was used as a standard.
Figure 1.—The IAA biosynthetic pathway identified in this study (in boldface type) and the analogous pathway identified
in U. maydis (right, underlined) where the homologs of Ald2 and Ald3 have been shown to catalyze the conversion of indole-
3-acetaldehyde to indole-3-acetic acid.
212R. P. Rao et al.
To screen the yeast deletion set, this assay was adapted for use
in 96-well microtiter dishes by scaling down the reaction
volume to 50 ml containing 0.1 mCi [14C]Trp.
Quantification of IAA from yeast: To confirm that IAA was
present in the CM, 5-ml cultures were harvested and stored at
?80?. The supernatants were thawed on ice and 38.4 ng of
of 2-propanol was added as an internal standard. Additionally,
500 ml of 0.2 m imidazole (pH 7.0) was added. The sample was
mixed and left to equilibrate on ice for 1 hr. The sample was
loaded onto a 200-mg NH2 solid phase extraction (SPE)
column (aka amino columns, Alltech) that was precondi-
tioned with sequential applications of 2 ml each hexane,
acetonitrile, and water and 0.2 m imidazole (pH 7.0) followed
by 6 ml of water on a vacuum manifold (Fisher Scientific,
Pittsburgh, PA). After loading the sample, the column was
5 psi. Next, the column was washed with sequential additions
of 1 ml each of hexane, ethyl acetate, acetonitrile, and
methanol. IAA was eluted in ?6 ml of methanol that was 5%
acetic acid. Dried samples were resuspended in 1.3 ml of a
mixture (?6:1 to reach a pH between 3 and 3.5) of 0.25%
phosphoric acid and 0.1 m succinic acid, pH 6.0. The sample
was placed in a 2-ml capacity 96-well plate and subjected to an
additional SPE step with polymethymethacrylate epoxide
resin, using a Gilson 215 SPE automated liquid handler
(ALH) as described in Barkawi et al. (2008). The epoxide SPE
column eluate was transferred to 2-ml amber vials, and ?1 ml
of ethereal diazomethane (prepared as described in Cohen
the sample was dried to a residue under a stream of N2gas in a
45? sand bath. The methylated IAA was resuspended in 45 ml
of ethyl acetate and subjected to GC-MS analysis as described
in Barkawi et al. (2008), except that a full scan spectrum was
obtained. For mutant analysis this protocol was scaled down to
1-ml cultures containing the same amount of [13C6]IAA
internal standard but only 0.2 ml of 0.2 m imidazole, pH 7.0.
S. cerevisiae secretes IAA: To confirm that S. cerevisiae
synthesizes IAA, we analyzed CM from yeast cultures.
Thin layer chromatography of CM from S. cerevisiae
grown in the presence of [14C]Trp revealed a radio-
labeled product that comigrated with commercially
available [14C]IAA (Figure 2A). UV shadow of the
fluor-impregnated TLC plate showed a UV absorbing
compound with the same retention profile as the pure
unlabeled IAA that was used as a standard (data not
shown but position marked with asterisk in Figure 2A).
GC-MS analysis of IAA that was extracted from the CM
along with [13C6]IAA internal standard and methylated
for GC analysis (Figure 2, B–D) confirmed the presence
of IAA in the CM. Figure 2B (left) shows the total ion
chromatogram (TIC) of pure methyl (Me)-IAA, which
shows a GC retention time for authentic Me-IAA to be
between 7.322 and 7.380 min. Figure 2B (right) shows
the full scan spectrum corresponding to this retention
ular ion) and mass to charge ration (m/z) 130 (fragment
consistent with pure Me-IAA as well, but were lower in
abundance and not typically used for quantification.
Figure 2C (bottom) shows the molecular and fragment
ions for Me-[13C6]IAA. Figure 2D (left) shows the TIC of
IAA that was extracted from 5 ml of CM (to which
[13C6]IAA had been added), methylated, and run on GC-
MS. The four predominant ions for Me-IAA and Me-[13-
spectrum for the retention time that corresponds to
authentic Me-IAA, demonstrating that yeast secretes IAA.
The accumulation of IAA in the CM reached its
highest level after cultures entered stationary phase.
To correlate the production of IAA with cell density,
cells from a high-density culture (108cells/ml) were
diluted to either low (5 3 105cells/ml) or high density
(5 3 107cells/ml) in fresh medium. IAA secreted into
Strains used in this study
Strain Description Source
cph1D/D efg1D/ D
MATa; his3D1; leu2D0; met15D0; ura3D0
ald2D in BY4741
ald3D in BY4741
ald4D in BY4741
ald5D in BY4741
ald6D in BY4741
ald2D ald3D in BY4741
ald2D ald3D in S1278b, MATa/a
Candida albicans wild type
Homozygous cph1D in Caf2-1
Homozygous efg1D in Caf2-1
Homozygous cph1D efg1D in Caf2-1
Heterozygous cap1D in Caf2-1
Homozygous cap1D in Caf2-1
J. Heitman, Duke University
G. Fink, MIT
G. Fink, MIT
G. Fink, MIT
G. Fink, MIT
M. Raymond, University of Montreal
M. Raymond, University of Montreal
aYeast Deletion Library.
IAA Synthesis in Yeast213
the medium was assessed by TLC (Figure 2E). After
normalizing for thedifference in cell number, we found
that CM taken from a high-density culture contained
more IAA than CM from a low-density culture (Figure
2E), indicating that IAA accumulation is directly pro-
portional to cell density. In S. cerevisiae, IAA is perceived,
and haploid invasive growth by regulating the cell sur-
face glycoprotein Flo11. Together these studies suggest
that IAA accumulates in the growth environment of
yeast where it may act as a chemical signal that regulates
A genomic scale screen for IAA homeostasis mu-
tants: To identify genes involved in IAA synthesis,
specifically the conversion of Trp to IAA, we initiated
an unbiased, systematic genomic screen of the yeast
1999). The haploid deletion library in S. cerevisiae
gene disruption. A [14C]Trp incorporation assay was
Figure 2.—(A) S. cerevisiae produces a molecule that comigrates with commercially available IAA. Wild-type yeast cells were
incubated with [14C]Trp, and products of the conditioned media were resolved by thin layer chromatography (TLC). Commer-
cially available IAA and [14C]IAA were used as controls. The position (marked with the asterisk) of the nonradiolabeled IAA con-
trol was determined by UV shadowing. (B) Total ion chromatogram (TIC, left) and full scan spectrum (right) of authentic methyl-
IAA. (C) Top, methyl-IAA molecular ion m/z 189 and fragment ion m/z 130 (the site of fragmentation to form the fragment ion is
indicated by dashed lines). Bottom, methyl-[13C6]IAA molecular ion m/z 195 and fragment ion m/z 136. For each compound, the
derivatization moiety (the methyl group) is shown in red. (D) TIC (left) and corresponding full-scan spectrum (right) of IAA
(methylated prior to GC-MS analysis) that was purified from the culture medium of wild-type yeast that had been grown in
the presence of Trp. The TIC shows four selected ions; m/z 130 and m/z 189 are the fragment ion and the molecular ion, re-
spectively, of endogenous IAA (methylated prior to GC-MS analysis). Ions with m/z 136 and 195 are the fragment ion and the
molecular ion, respectively, of [13C6]IAA (methylated prior to GC-MS analysis) that was added to the yeast culture medium su-
pernatant prior to extraction of IAA. The large peak in the TIC (left) with a retention time of ?7.15 min was determined to
be tryptophol by full-scan spectra analysis (not shown). (E) The CM taken from a high-density culture contained a much greater
concentration of IAA than CM from a low-density culture as determined by TLC (bottom) and densitometry of the autoradio-
214 R. P. Rao et al.
developed and optimized to facilitate a large-scale
screen using microtiter plates. An aliquot of the CM
from each reaction was loaded onto a TLC plate and
components of the CM were resolved and compared
with a14C-IAA standard. A total of 1425 deletion strains
(29% of the library) have been screened to date. A
secondary screen was performed in triplicate on puta-
tive mutants and related gene families, using the
[14C]Trp incorporation assay.
This screen identified three genes, ALD2, ARO9, and
ADH2, representing families of particular interest with
respect to IAA biosynthesis in yeast: the ALDehyde
dehydrogenases, the AROmatic transaminases, and
the Alcohol DeHydrogenases (Figure 1). In S. cerevisiae,
the aromatic transaminases Aro8 and Aro9 have been
implicated in the conversion of Trp to indole pyruvate
(IPA) (Chen and Fink 2006). As expected, aro8D and
aro9D mutants show decreased conversion of labeled
Trp to labeled IAA compared to the cognate wild type
but are not the focus of this study (data not shown).
acetaldehyde (IAAld) to indole-3-ethanol (aka trypto-
phol) (Chen and Fink 2006). Interestingly, adh2D,
identified in the screen, was the only member of the
ADH family to show decreased [14C]IAA accumulation
(data not shown). One explanation for this result is that
Adh2 preferentially catalyzes the conversion of ethanol
to acetylaldehyde. Therefore adh2D mutants are unable
to convert indole-3-ethanol to IAAld, ultimately leading
to decreased IAA accumulation. Deletion mutants of
members of the ALD family accumulated lower levels of
radioactive IAA from radioactive Trp than did wild type.
We focused our study on the aldehyde dehydrogenase
(ALD) genes hypothesized to catalyze the ultimate step
in the production of IAA and set out to test whether
altering IAA production affects filamentation. Multiple
shown) indicate that S. cerevisiae Ald2 and Ald3 share
identity with U. maydis Iad1. Ald2 and Ald3 are nearly
identical to each other and have 50% (Ald3) and 49%
(Ald2) protein sequence identity with U. maydis Iad1, a
have less sequence identity with the NADH-dependent
Single and double deletions of the ALD genes showed
decreased IAA production from [14C]Trp when com-
pared with wild-type cells on TLC (Figure 3). These
results together with previous enzymatic studies in U.
maydis (Reineke et al. 2008) suggest that these genes are
involved in IAA synthesis. ALD2 and ALD3 are also
required for synthesis of a nonproteinogenic amino
acid, b-alanine in S. cerevisiae (White et al. 2003).
The ald2Dald3D deletion mutant exhibits virulence
traits: IAA regulates dimorphic transition in S. cerevisiae
by inducing adhesion and filamentation (Prusty et al.
2004). The ability of a fungus to perceive a small mo-
form has important implications for host–pathogen
interactions. To test the hypothesis that mutants with
aberrant IAA accumulation also affect dimorphism, we
examined diploid filamentation and haploid invasive
growth in all ald single mutants and selected combina-
tions of double mutants. The ald2Dald3D double mu-
tant demonstrated increased filamentation (Figure 4A)
and invasive growth (Figure 4B) as compared wild type.
We also tested a previously reported growth inhibition
This IAA-associated growth inhibition phenotype ex-
hibits a direct proportionality between IAA concentra-
tion and growth inhibition. Deletion of both ald2 and
ald3 caused an increase in sensitivity to IAA (Figure 4C,
right) whereas single deletion of an ALD gene did not
affect IAA sensitivitywhen comparedwith wild-typecells
(data not shown). Together, these data suggest that a
adhesion and filamentation of S. cerevisiae. However,
these phenotypes are consistent with ald2Dald3D mu-
tants producing more IAA than isogenic wild-type
The ald2Dald3D mutant uncovers an IAA biosyn-
thetic pathway that is independent of exogenous Trp:
The ALD genes were identified on the basis of a
radiolabeled [14C]Trp incorporation assay. IAA accumu-
lation in the CM of the double mutant was quantified
using GC-MS and [13C6]IAA as an internal standard.
These measurements revealed that the CM of the
ald2Dald3D deletion mutant contained fourfold more
IAA (240.3 ng/ml 6 71.9 ng/ml) than the wild type
(59.8 ng/ml 6 3.8 ng/ml). The amount of IAA present
in the conditioned media is adequate to induce fila-
mentation in an in vitro plate assay. Together these
analytical data correlate well with the phenotypic data,
suggesting that the ald2Dald3D double mutant makes
more IAA and thus exhibits enhanced virulence traits as
compared to its wild-type counterpart.
While the radiolabeled [14C]Trp incorporation assay
detects the pool of IAA synthesized from labeled Trp,
Figure 3.—Products of the CM of ald single deletion mu-
tants and ald2Dald3D double deletion incubated with
[14C]Trp were resolved by TLC and compared to the iso-
genic wild-type strain. Each experiment was performed a
minimum of three times. Three and two independent trans-
formants were tested for the single and double mutants, re-
spectively. One representative transformant for each mutant
IAA Synthesis in Yeast215
the GC-MS analysis allowed us to detect any unlabeled
(endogenous) IAA that was present. We grew the
ald2Dald3D double mutant in the absence of exogenous
Trp and quantified IAA from the CM using GC-MS.
These measurements revealed that the ald2Dald3D
mutant was able to synthesize a modest amount of IAA
(9.48 ng/ml 6 0.22 ng/ml) in the absence of exogenous
in the absence of Trp (9.81 ng/ml 6 0.77 ng/ml).
IAA induces filamentation in C. albicans: The effects
of the secondary metabolites identified in fungi appear
to be largely species specific (Chen and Fink 2006).
Previous work suggests that IAA induces invasive growth
in S. cerevisiae (Prusty et al. 2004). To test whether
the IAA effects could cross species barriers, we exposed
wild-type Candida albicans, a human pathogen, as well
as attenuated mutants in the mitogen-activated pro-
tein (MAP) kinase and the cAMP-dependent protein
kinase pathways (Figure 5) to IAA. The cph1D/ D efg1D/D
double mutant, which fails to switch from the vegeta-
tive to the filamentous form, was filamentous in the
presence of IAA (compare Figure 5A with 5E). The
single mutants efg1D/D or cph1D/D that normally show
reduced filamentation also showed a robust filamenta-
tion when exposed to IAA (compare Figure 5B with 5F
and 5C with 5G). Wild-type strains also filamented more
when treated with IAA as compared to untreated cells
(compare Figure 5D and 5H). These results indicate
that IAA enhances filamentation of the human patho-
gen C. albicans. Furthermore, the IAA-mediated fila-
mentation signal does not require components of
the MAPK or PKA pathways. The cph1D/D efg1D/ D
double mutant, which is nonfilamentous under stan-
dard laboratory conditions and avirulent in mice,
and when embedded in agar (Riggle et al. 1999).
Together these results suggest that IAA-mediated fila-
independent mechanism and confirm prior findings
were also conserved in C. albicans, we tested Cap1, the
C. albicans homolog of Yap, for its sensitivity to IAA. The
amino acid auxin permeases genes are upregulated in
the yap1 mutant, which is sensitive to growth on IAA
because it retains more IAA (Prusty et al. 2004).
Heterozygous and homozygous deletion mutants of
CAP1 (Alarco and Raymond 1999) (obtained from
M. Raymond, University of Montreal) to grew less well
on media containing IAA as compared to the isogenic
wild type (Figure 5I), suggesting that the cap1D/D
mutant was more sensitive to IAA. The heterozygous
mutant, cap1D/1 exhibited an intermediate sensitivity
to IAA as compared to the wild-type CAP11/1 strain or
the homozygous cap1D/D deletion strain. These results
suggest that cap1 mutants are hypersensitive to IAA,
further supporting our hypothesis that the molecular
Figure 4.—(A) Arepresentativediploidald2D/D
ald3D/D colony was grown on filamentation-
inducing media and photographed after 3 days
of growth (bar, 1 mm). (B) Haploid ald2Dald3D
strains were spotted onto SC media and washed.
Before wash, unwashed plates; after wash, the
plates after washing. (C) A filter disk saturated
with IAA (right) was placed on a lawn of
ald2Dald3D mutant cells (bottom) and compared
tothewild-type cells(top).Control disks(left)do
not contain IAA. Plates were incubated for 3 days
in the dark. The clear area around the IAA-
containing filter disks indicates a zone of growth
216R. P. Rao et al.
mechanism of IAA response is likely to be conserved
between S. cerevisiae and C. albicans.
The quantitative GC-MS analysis in this study con-
firmed that S. cerevisiae synthesizes and secretes IAA into
as a signal that regulates filamentation. Filamentation is
a pathogenic trait because it contributes directly to
virulence of pathogenic fungi like C. albicans. Patho-
genic bacteria and fungi are known to produce IAA, but
a direct link to pathogenicity has not been demon-
strated in these pathogens.
IAA is a small molecule capable of stimulating the
developmentaltransition from the vegetativeyeast form
to the filamentous form in S. cerevisiae (Prusty et al.
2004). The current study provides strong support for a
connection between fungal dimorphism and IAA syn-
thesis, because the ald2Dald3D strain that accumulates
more IAA is also more filamentous. IAA was also able to
stimulate dimorphic transition in the human pathogen
C. albicans. Deletion of a key regulator of the IAA
responses had the same effect in both organisms.
Homologs of enzymes that transport and synthesize
IAA in S. cerevisiae are present in C. albicans. We suggest
that IAA is an important signal that triggers dimorphic
transition—a virulence trait.
A genomic scale screen for IAA homeostasis mutants
implicated the aldehyde dehydrogenases, Ald2 and
Ald3 in the final step of IAA synthesis from Trp. Ald2
and Ald3 share significant sequence similarity with
Iad1, the U. maydis aldehyde dehydrogenase that has
been shown to catalyze the conversion of IAAld to IAA
(Basse et al. 1996; Akamatsu et al. 2000; Mizuno et al.
ALD genes are responsible for acetate formation during
anaerobic fermentation (Saint-Prix et al. 2004; Pigeau
and Inglis 2007) and are hence of interest to the
brewing industry. They have previously been implicated
in mediating a variety of stress responses and are
regulated by general-stress transcription factors Msn2
and -4 (Miralles and Serrano 1995; Navarro-Avino
et al. 1999; Aranda and del Olmo 2003). Ald activity is
required in the synthesis of two amino acid derivatives,
IAA and b-alanine in U. maydis and S. cerevisiae, re-
spectively (White et al. 2003; Reineke et al. 2008). This
which has previously been implicated in the first step of
IAA synthesis (Chen and Fink 2006). In the process of
characterizing mutants in a Trp-dependent IAA synthe-
sis pathway, we uncovered another pathway that did not
rely on exogenous Trp for IAA biosynthesis. Trp-
independent synthesis of IAA has been demonstrated
in several plant species, but the intermediates, interme-
diate steps, and genes involved in this pathway remain
undefined (Woodward and Bartel 2005; Normanly
2009). The observation that S. cerevisiae has an analo-
in the characterization of Trp-independent IAA synthesis.
Figure 5.—The human
pathogen Candida albicans
was exposed to IAA [exper-
imental plates (E–H) con-
no IAA; bar in A, 10 mm].
A–H show the edge of a
patch of C. albicans (A
and E, cph1efg1; B and F,
cph1; C and G, efg1; and D
and H, isogenic wild-type
on synthetic low ammo-
nium media with xylose as
a carbon source. Plates
were incubated in the dark
to prevent photodegrada-
tion of IAA. I and J show
the IAA sensitivity profile
of a cap1 homozygous dele-
tion mutant as compared
to an isogenic wild-type
and a heterozygous mutant
contain 120 mm IAA, and
control plates (J) contain
IAA Synthesis in Yeast217
There is precedence for multiple IAA biosynthetic
pathways in microbes, particularly plant-associated bac-
1995; Glick et al. 1999b; Lambrecht et al. 2000). An
interesting example of differential utilization of multi-
ple IAA biosynthetic pathways in microbes is found in
Erwinia herbicola, which requires a functional indole
acetamide (IAM) pathway (Trp is converted to IAM and
then to IAA) to be pathogenic to plants and requires a
functional IPA pathway (Figure 1) to exist as a plant
epiphyte (Manulis et al. 1998). We note that while al-
dehyde dehydrogenase has been implicated in IAA
synthesis in U. maydis, this pathway is not involved in
tumorigenesis (Reineke et al. 2008). This result is con-
necessary for IAA-induced filamentation and that an
alternate IAA synthesis pathway likely exists in yeast.
The coexistence of both Trp-dependent and Trp-
independent IAA-biosynthetic pathways has been docu-
mented in plants (Normanly et al. 2004; Woodward
and Bartel 2005) and microbes (Prinsen et al. 1993).
In plants, Trp-independent IAA synthesis is proposed to
both precursors of Trp (Normanly et al. 2004). One of
for plants converts Trp to IPA (reviewed in Woodward
and Bartel 2005 and Normanly 2009). The Arabidop-
sis TAA-1 protein can convert Trp to IPA in vitro, and
mutations in theTAA-1 gene produce less IAA when the
plant is subjected to simulated shade (Tao et al. 2008),
high temperature (Yamada et al. 2009), or ethylene
(Stepanova et al. 2008). IAAld has been proposed as an
intermediate of the Trp-dependent IAA synthetic path-
wayin plants, butthis has yet to be confirmed, and plant
orthologs of ALD genes have not been identified. One
putative aldehyde oxidase from Arabidopsis shows a
substratepreferencefor IAAldinvitro,but therelevance
of this gene to IAA biosynthesis in vivo has yet to be
confirmed (Seo et al. 1998). Future studies will involve
using differential stable isotope labeling coupled with
genetic mutants to identify components of alternate
IAA biosynthetic pathways in S. cerevisiae.
Secondary metabolites are recognized as important
signals. Aspergillus fumigatus hyphae release a small
molecule, gliotoxin, which can exacerbate the patho-
genesis of invasive aspergillosis (Sutton et al. 1996).
Pseudomonas aeruginosa produces a signaling molecule,
homoserine lactone, which inhibits C. albicans filamen-
tation (Hogan et al. 2004). Two predominant types of
small molecules, acyl homoserine lactones (AHLs)
(Fuqua et al. 2001; Danhorn et al. 2004; Akimkina
et al. 2006) and modified oligopeptides (Kleerebezem
bacteria, respectively, to regulate phenotypes that lead
to virulence such as antibiotic production and biofilm
formation. C. albicans has been shown to produce
secondary metabolites such as tyrosol and farnesol that
regulate dimorphic transition (Shchepin et al. 2003;
Chen et al. 2004). Aromatic alcohols such as tryptophol
and phenylalanol, a catabolic product of Phe, are
produced by both S. cerevisiae and C. albicans but exert dif-
ferent effects on their morphogenesis, suggesting that
they have distinct species-specific effects. IAA differs
from these previously described signaling molecules
because its effects appear to cross species barriers.
Diverse fungal species respond to IAA; therefore, de-
fining the pathways by which IAA regulates filamenta-
tion in C. albicans will yield a better understanding of its
spectrum antifungal therapies. Furthermore, auxin
permeases that import IAA in S. cerevisiae are homolo-
gous to the Arabidopsis IAA importer, Aux1 (Prusty
et al. 2004). Therefore, defining IAA synthesis and
regulation in yeast, a simple eukaryote, will yield a
better understanding of IAA regulation in plants.
The authors thank G. Fink, J. Celenza, M. Lorenz, and J. Cohen for
critical reading of the manuscript and helpful discussions and
acknowledge C. Jain and M. Lewandowski for assistance with sample
in part by Worcester Polytechnic Institute and National Science
Foundation funds MCB 0517420 to J.N.
Abel, S., and T. Athanosios, 2010
Harbor Perspectives in Biology. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Akamatsu, S., H. Kamiya, N. Yamashita, T. Motoyoshi, N.
Goto-Yamamoto et al., 2000
nase and acetyl-CoA synthetase on acetate formation in sake
mash. J. Biosci. Bioeng. 90: 555–560.
Akimkina, T., K. Yook, S. Curnock and J. Hodgkin, 2006
characterization, analysis of virulence and transformation of
Microbacterium nematophilum, a coryneform pathogen of the
nematode Caenorhabditis elegans. FEMS Microbiol. Lett. 264:
Alarco, A. M., and M. Raymond, 1999
tor Cap1p is involved in multidrug resistance and oxidative stress
response in Candida albicans. J. Bacteriol. 181: 700–708.
Aranda, A., and M. L. del Olmo, 2003
stress in the yeast Saccharomyces cerevisiae involves a strain-dependent
regulation of several ALD genes and is mediated by the general
stress response pathway. Yeast 20: 747–759.
Barash, I., and S. Manulis-Sasson, 2009
terial pathogens: thegall-forming Pantoeaagglomeranscase. Annu.
Rev. Phytopathol. 47: 133–152.
Barkawi, L. S., Y. Y. Tam, J. A. Tillman, B. Pederson, J. Calio et al.,
2008A high-throughput method for the quantitative analysis of
indole-3-acetic acid and other auxins from plant tissue. Anal. Bio-
chem. 372: 177–188.
Basse, C. W., F. Lottspeich, W. Steglich and R. Kahmann,
1996Two potential indole-3-acetaldehyde dehydrogenases in
the phytopathogenic fungus Ustilago maydis. Eur. J. Biochem.
Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li et al.,
1998 Designer deletion strains derived from Saccharomyces cere-
visiae S288C: a useful set of strains and plasmids for PCR-mediated
gene disruption and other applications. Yeast 14: 115–132.
Brown, Jr., D. H., A. D. Giusani, X. Chen and C. A. Kumamoto,
1999Filamentous growth of Candida albicans in response to
physical environmental cues and its regulation by the unique
CZF1 gene. Mol. Microbiol. 34: 651–662.
Chen, H., and G. R. Fink, 2006 Feedback control of morphogenesis
in fungi by aromatic alcohols. Genes Dev. 20: 1150–1161.
Odyssey of auxin, in Cold Spring
Effects of aldehyde dehydroge-
The bZip transcription fac-
Response to acetaldehyde
Recent evolution of bac-
218 R. P. Rao et al.
Chen, H., M. Fujita, Q. Feng, J. Clardy and G. R. Fink,
2004 Tyrosol is a quorum-sensing molecule in Candida albicans.
Proc. Natl. Acad. Sci. USA 101: 5048–5052.
Clark, E., S. Manulis, Y. Ophir, I. Barash and Y. Gafni, 1993
ing and Characterization of iaaM and iaaH from Erwinia herbicola
pathovar gypsophilae. Mol. Plant Pathol. 83: 234–240.
Cohen, J. D., 1984 Convenient apparatus for the generation of
small amounts of diazomethane. J. Chromatogr. 303: 193–196.
Costacurta, A., and J. Vanderleyden, 1995
mones by plant-associated bacteria. Crit. Rev. Microbiol. 21: 1–18.
Danhorn, T., M. Hentzer, M. Givskov, M. R. Parsek and C. Fuqua,
2004 Phosphorus limitation enhances biofilm formation of
the plant pathogen Agrobacterium tumefaciens through the PhoR-
PhoB regulatory system. J. Bacteriol. 186: 4492–4501.
Delker, C., A. Raschke and M. Quint, 2008
dazzling complexity of a small molecule’s message. Planta 227:
Dieterich, C., M. Schandar, M. Noll, F. J. Johannes, H. Brunner
et al., 2002 In vitro reconstructed human epithelia reveal con-
tributions of Candida albicans EFG1 and CPH1 to adhesion and
invasion. Microbiology 148: 497–506.
Fuqua, C., M. R. Parsek and E. P. Greenberg, 2001
gene expression by cell-to-cell communication: acyl-homoserine
lactone quorum sensing. Annu. Rev. Genet. 35: 439–468.
Furukawa, T., J. Koga, T. Adachi, K. Kishi and K. Syono,
1996 Efficient conversion of L-tryptophan to indole-3-acetic
acid and/or tryptophol by some species of Rhizoctonia. Plant Cell
Physiol. 37: 899–905.
Glick, B. R., C. L. Patten, G. Holguin and D. M. Penrose,
1999a Auxin production, pp. 86–133 in Biochemical and Genetic
Mechanisms Used by Plant Growth Promoting Bacteria, edited by B. R.
Glick, C. L. Patten, G. Holguin and D. M. Penrose. Imperial
College Press, London.
Glick, B. R., C. L. Patten, G. Holguin and D. M. Penrose,
Biochemical and Genetic Mechanisms Used by Plant Growth
Promoting Bacteria. Imperial College Press, London.
Gruen, H. E., 1959 Auxins and fungi. Annu. Rev. Plant Physiol. 10:
Guthrie, C., and G. R. Fink, 1991
lecular biology. Methods Enzymol. 194: 1–863.
Halliday, K. J., J. F. Martinez-Garcia and E.-M. Josse, 2009
tegration of light and auxin signaling. Cold Spring Harb. Per-
spect. Biol. (in press).
Hazelwood, L. A., J. M. Daran, A. J. van Maris, J. T. Pronk and J. R.
Dickinson, 2008The Ehrlich pathway for fusel alcohol
production: a century of research on Saccharomyces cerevisiae
metabolism. Appl. Environ. Microbiol. 74: 2259–2266.
Hirsch, A. M., T. V. Bhuvaneswari, J. G. Torrey and T. Bisseling,
1989Early nodulin genes are induced in alfalfa root out-
growths elicited by auxin transport inhibitors. Proc. Natl. Acad.
Sci. USA 86: 1244–1248.
Hogan, D. A., A. Vik and R. Kolter, 2004
quorum-sensing molecule influences Candida albicans morphol-
ogy. Mol. Microbiol. 54: 1212–1223.
Kleerebezem, M., L. E. Quadri, O. P. Kuipers and W. M. de Vos,
1997 Quorum sensing by peptide pheromones and two-
component signal-transduction systems in Gram-positive bacte-
ria. Mol. Microbiol. 24: 895–904.
Lambrecht, M., Y. Okon, A. Vande Broek and J. Vanderleyden,
2000Indole-3-acetic acid: a reciprocal signalling molecule in
bacteria-plant interactions. Trends Microbiol. 8: 298–300.
Lau, S., G. Jurgens and I. De Smet, 2008
of the auxin pathway. Plant Cell 20: 1738–1746.
Liu, H., 2001Transcriptional control of dimorphism in Candida al-
bicans. Curr. Opin. Microbiol. 4: 728–735.
Lo, H. J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A.
Cacciapuoti et al., 1997Nonfilamentous C. albicans mutants
are avirulent. Cell 90: 939–949.
Manulis, S., A. Haviv-Chesner, M. T. Brandl, S. E. Lindow and I.
Barash, 1998Differential involvement of indole-3-acetic acid
biosynthetic pathways in pathogenicity and epiphytic fitness of
Erwinia herbicola pv. gypsophilae. Mol. Plant-Microbe Interact.
McSteen, P., 2010Auxin and monocot development. Cold Spring
Harb. Perspect. Biol. (in press).
Synthesis of phytohor-
Auxin dynamics: the
Guide to yeast genetics and mo-
A Pseudomonas aeruginosa
The evolving complexity
Miralles, V. J., and R. Serrano, 1995
myces cerevisiae with four genes up-regulated by osmotic stress.
Mol. Microbiol. 17: 653–662.
Mizuno, A., H. Tabei and M. Iwahuti, 2006
low-acetic-acid-producing yeast isolated from 2-deoxyglucose-
resistant mutants and its application to high-gravity brewing.
J. Biosci. Bioeng. 101: 31–37.
Moller, B., and D. Weijers, 2009
ing. Cold Spring Harb. Perspect. Biol. (in press).
Navarro-Avino, J. P., R. Prasad, V. J. Miralles, R. M. Benito and
R. Serrano, 1999A proposal for nomenclature of aldehyde
dehydrogenases in Saccharomyces cerevisiae and characterization
of the stress-inducible ALD2 and ALD3 genes. Yeast 15: 829–842.
Nielsen, N., 1931U ¨ber Wuchsstoffe der Hefe. Biochem. Z. 237:
Normanly, J., 2009
Approaching Cellular and Molecular Resolution of
Auxin Biosynthesis and Metabolism. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Normanly, J., J. Sovin and J. Cohen, 2004
Hormones: Biosynthesis, Signal Transduction, Action. Kluwer Aca-
demic Publishers, Dordrecht, The Netherlands.
Pigeau, G. M., and D. L. Inglis, 2007
charomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde
stress during icewine fermentation. J. Appl. Microbiol. 103:
Prinsen, E., A. Costacura, K. Michiels, J. Vanderleyden and H.
Van Onckelen, 1993
Azospirillum brasilense indole-3-acetic acid
biosynthesis: evidence for a non-tryptophan-dependent pathway.
Mol. Plant-Microbe Interact. 6: 609–615.
Prusty, R., P. Grisafi and G. R. Fink, 2004
doleacetic acid induces invasive growth in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA 101: 4153–4157.
Reineke, G., B. Heinze, J. Schirawski, H. Buettner, R. Kahmann
et al., 2008 Indole-3-acetic acid (IAA) biosynthesis in the smut
fungusUstilagomaydisand its relevance for increasedIAA levelsin
infected tissue and host tumour formation. Mol. Plant Pathol. 9:
Reneke, J. E., K. J. Blumer, W. E. Courchesne and J. Thorner,
1988 The carboxy-terminal segmentof the yeast alpha-factor re-
ceptor is a regulatory domain. Cell 55: 221–234.
Riggle, P. J., K. A. Andrutis, X. Chen, S. R. Tzipori and C. A.
Kumamoto, 1999Invasive lesions containing filamentous
forms produced by a Candida albicans mutant that is defective
in filamentous growth in culture. Infect. Immun. 67: 3649–3652.
Robinson, M., J. Riov and A. Sharon, 1998
synthesis in Colletotrichum gloeosporioides f. sp. aeschynomene. Appl.
Environ. Microbiol. 64: 5030–5032.
Saint-Prix, F., L. Bonquist and S. Dequin, 2004
ysis of the ALD gene family of Saccharomyces cerevisiae during
anaerobic growth on glucose: the NADP1-dependent Ald6p
and Ald5p isoforms play a major role in acetate formation. Micro-
biology 150: 2209–2220.
Scarpella, E., M. Barkoulas and M. Tsiantis, 2010
leaf and vein development by auxin. Cold Spring Harb. Perspect.
Biol. (in press).
Seo, M., S. Akaba, T. Oritani, M. Delarue, C. Bellini et al.,
1998Higher activity of an aldehyde oxidase in the auxin-
overproducing superroot1 mutant of Arabidopsis thaliana. Plant
Physiol. 116: 687–693.
Shchepin, R., J. M. Hornby, E. Burger, T. Niessen, P. Dussault
et al., 2003Quorum sensing in Candida albicans: probing farne-
sol’s mode of action with 40 natural and synthetic farnesol ana-
logs. Chem. Biol. 10: 743–750.
Sherman, F., G. R. Fink and J. B. Hicks, 1986
ual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Shin, M., T. Shinguu, K. Sano and C. Umezawa, 1991
fates of L-tryptophan in Saccharomyces uvarum (Saccharomyces
carlsbergensis). Chem. Pharm. Bull. (Tokyo) 39: 1792–1795.
Sohn, K., C. Urban, H. Brunner and S. Rupp, 2003
regulator of cell wall dynamics in Candida albicans as revealed by
DNA microarrays. Mol. Microbiol. 47: 89–102.
Stepanova, A. N., J. Robertson-Hoyt, J. Yun, L. M. Benavente, D. Y.
Xie et al., 2008
TAA1-mediated auxin biosynthesis is essential for
hormone crosstalk and plant development. Cell 133: 177–191.
A genomic locus in Saccharo-
Auxin control of embryo pattern-
Auxin Metabolism in Plant
Response of wine yeast (Sac-
The plant hormone in-
Indole-3-acetic acid bio-
Laboratory Course Man-
EFG1 is a major
IAA Synthesis in Yeast219
Stewart, J. L., and J. L. Nemhauser, 2009
money? Auxin as the currency of the cellular economy. Cold
Spring Harb. Perspect. Biol. (in press).
Sundberg, E., and L. Ostergaard, 2009
auxin activities during reproductive development. Cold Spring
Harb. Perspect. Biol. (in press).
Sutton, P., P. Waring and A. Mullbacher, 1996
invasive aspergillosis by the immunosuppressive fungal metabo-
lite, gliotoxin. Immunol. Cell. Biol. 74: 318–322.
Tao, Y., J. L. Ferrer, K. Ljung, F. Pojer, F. Hong et al., 2008
synthesis of auxin via a new tryptophan-dependent pathway is re-
quired for shade avoidance in plants. Cell 133: 164–176.
Thimann, K. V., 1935 On the plant growth hormone produced by
Rhizopus suinus. J. Biol. Chem. 109: 279–291.
Turian, G., and R. H. Hamilton, 1960
3-indolylacetic acid in Ustilago zeae tumors. Biochim. Biophys.
Acta 41: 148–150.
Viglierchio, D. R., 1971 Nematodes and other pathogens in auxin-
related plant-growth disorders. Bot. Rev. 37: 1–21.
Wach, A., A. Brachat, R. Pohlmann and P. Philippsen, 1994
heterologous modules for classical or PCR-based gene disrup-
tions in Saccharomyces cerevisiae. Yeast 10: 1793–1808.
Do trees grow on
Distinct and dynamic
Chemical detection of
White, W. H., P. L. Skatrud, Z. Xue and J. H. Toyn,
2003 Specialization of function among aldehyde dehy-
drogenases: the ALD2 and ALD3 genes are required for
beta-alanine biosynthesis in Saccharomyces cerevisiae. Genetics
Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K.
Anderson et al., 1999Functional characterization of the S.
cerevisiae genome by gene deletion and parallel analysis. Science
Woodward, A. W., and B. Bartel, 2005
and interaction. Ann. Bot. (Lond) 95: 707–735.
Yamada, M., K. Greenham, M. J. Prigge, P. J. Jensen and M. Estelle,
2009 The transport inhibitor response2 (TIR2) gene is
required for auxin synthesis and diverse aspects of plant develo-
pment. Plant Physiol. (in press).
Zazimalova, E., A. S. Murphy, K. Hoyerova and P. Hosek,
2009Auxin transporters—Why so many? Cold Spring Harb.
Perspect. Biol. (in press).
Auxin: regulation, action,
Communicating editor: A. P. Mitchell
220 R. P. Rao et al.