RESEARCH ARTICLE

Life without putrescine: disruption of the gene-encoding

polyamine oxidase in Ustilago maydis odc mutants
Laura Valdés-Santiago1, Doralinda Guzmán-de-Peña2 & José Ruiz-Herrera1
1
Departamento de Ingenierı́a Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato,
Guanajuato, Mexico; and 2Departamento de Bioquı́mica y Biotecnologı́a, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico

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Correspondence: José Ruiz-Herrera,
Departamento de Ingenierı́a Genética,
Unidad Irapuato, Centro de Investigación y de
Estudios Avanzados del Instituto Politécnico
Nacional, Apartado Postal 629, 36821
Irapuato, Guanajuato, Mexico. Tel.: 152 462
623 9600; fax: 152 462 624 5849;
e-mail: jruiz@ira.cinvestav.mx
Received 20 May 2010; revised 8 July 2010;
accepted 21 July 2010.
Final version published online 13 September
2010.
DOI:10.1111/j.1567-1364.2010.00675.x
Editor: Richard Calderone
Keywords
polyamines; polyamine oxidase; Ustilago
maydis; virulence; stress response.
Abstract
In previous communications the essential role of spermidine in Ustilago maydis
was demonstrated by means of the disruption of the genes encoding ornithine
decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of
specific roles to each polyamine in different cellular functions was not possible
because the spermidine added to satisfy the auxotrophic requirement of odc/spe
double mutants is partly back converted into putrescine. In this study, we have
approached this problem through the disruption of the gene-encoding polyamine
oxidase (PAO), required for the conversion of spermidine into putrescine, and the
construction of odc/pao double mutants that were unable to synthesize putrescine
by either ornithine decarboxylation or retroconversion from spermidine. Pheno-
typic analysis of the mutants provided evidence that putrescine is only an
intermediary in spermidine biosynthesis, and has no direct role in cell growth,
dimorphic transition, or any other vital function of U. maydis. Nevertheless, our
results show that putrescine may play a role in the protection of U. maydis against
salt and osmotic stress, and possibly virulence. Evidence was also obtained that the
retroconversion of spermidine into putrescine is not essential for U. maydis growth
but may be important for its survival under natural conditions.

models to study the physiological roles of these important

Introduction micromolecules (Balasundaram et al., 1991; Walters, 1995).
YEAST RESEARCH

In the last few decades, the study of polyamine metabolism
has attracted the attention of many groups interested in the
understanding of growth and differentiation phenomena in
different organisms (Tabor & Tabor, 1985; Cohen, 1998;
Morgan, 1999; Igarashi & Kashiwagi, 2000) because it is well
known that these molecules play a role in cell and tissue
proliferation, differentiation, and growth (Ruiz-Herrera,
1994; Auvinen et al., 1997; Farriol et al., 2001). Even though
polyamines are structurally simple, there is evidence of their
important role in a variety of biological processes such as cell
cycle regulation, gene expression, and signal transduction
(Patel & Wang, 1997; Ray et al., 1999; Bachrach et al., 2001;
Chattopadhyay et al., 2009; Koomoa et al., 2009).
The pathways of polyamine metabolism in plants and
animals have been extensively studied (e.g. Gerner & Meys-
kens, 2004; Casero & Marton, 2007; Efrose et al., 2008;
Kusano et al., 2008), but fungi may also constitute good
Our research group has obtained evidence of the direct
relationship between polyamine synthesis and differentia-
tion in a large number of fungal species by means of the use
of inhibitors of their synthesis (e.g. Calvo-Méndez et al.,
1987; Martı́nez-Pacheco et al., 1989; reviewed by Ruiz-
Herrera, 1994), and the isolation of mutants deficient in
the gene-encoding ornithine decarboxylase (ODC) (Gue-
vara-Olvera et al., 1997; Herrero et al., 1999; Jiménez-
Bremont et al., 2001). An attractive model for these studies
is the phytopathogenic basidiomycete Ustilago maydis,
which contains only two (putrescine and spermidine) of
the three widely distributed polyamines (putrescine, sper-
midine, and spermine), simplifying their study (Valdés-
Santiago et al., 2009).
One unsolved problem of polyamine functions concerns
the specific roles of each polyamine in cell growth and
differentiation. Previously, using single mutants in the


c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 928–940
Published by Blackwell Publishing Ltd. All rights reserved
Life without putrescine 929

gene-encoding spermidine synthase (spe), and double odc/
spe mutants, we obtained evidence that in U. maydis,
spermidine can fulfill all the functions ascribed to poly-
amines (Valdés-Santiago et al., 2009). Nevertheless, the
question of a specific role for putrescine remained unan-
swered, as those mutants still possessed the capacity to
synthesize putrescine by the retroconversion of spermidine
into putrescine. It is known that putrescine is not only
synthesized by decarboxylation of ornithine by Odc (Sebela
et al., 2001; Seiler, 2004), but also by the FAD-dependent
polyamine oxidase (Pao), which catalyzes the oxidation of
When necessary (see Results), media were supplemented
with one or more of the following compounds: hygromycin
(200 mg mL1), carboxin (20 mM), and spermidine or pu-
trescine in different concentrations. Yeast or mycelial growth
in liquid or solid media was obtained as described (Ruiz-
Herrera et al., 1995). Comparative growth of U. maydis
strains was measured on plates of solid media inoculated
with 10 mL drops of decimal dilutions from cell suspensions
containing 108 cells mL1. After 24 and 48 h of incubation at
28 1C, the relative growth was observed, and the plates were
photographed.

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N1-acetylspermidine to produce N-acetyl-3-aminopropa- Escherichia coli was grown at 37 1C in Luria–Bertani
naldehyde and putrescine (Pegg, 1988). In the present medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium
article, we describe the isolation and disruption of the chloride) with ampicillin (100 mg mL1) or kanamycin
gene-encoding Pao, and the phenotypic characteristics of (50 mg mL1) added for plasmid selection.
single pao and double pao/odc mutants, as an approach to
understand the specific roles of putrescine in U. maydis
growth and development. Techniques for nucleic acid manipulation
Genomic DNA from U. maydis was isolated by a glass bead
Materials and methods lysis method (Hoffman & Wriston, 1987). Total RNA was
isolated according to Jones et al. (1985). Southern and
Northern hybridizations of 10 and 30 mg of nucleic acid
Strains, culture media, and growing conditions
samples, respectively, were performed by standard techni-
The strains of U. maydis and Escherichia coli used in this ques (Sambrook & Rusell, 2001). DNA probes were labeled
study are described in Table 1. Ustilago maydis strains were using the random primer labeling system and [32P]a dCTP
maintained in 50% glycerol at  70 1C. When necessary, (Amersham Biosciences, Little Chalfont, UK).
they were recovered in complete medium (CM; Holliday, Plasmid DNA isolation from E. coli was performed by
1974) containing different additions (see Results), and standard procedures (Sambrook & Rusell, 2001). Plasmid
incubated for about 48 h at 28 1C under shaking conditions. isolation from U. maydis was performed according to
These cultures were used as inocula, normally for minimal Sobanski & Dickinson (1995).
medium (MM; Holliday, 1974), and incubated as above. DNA enzymatic reactions such as digestion, ligation, and
vector dephosphorylation were performed as recommended
by the manufacturers of the reagents used (Invitrogen,
Table 1. Strains used in this study Carlsbad, CA, and New England Biolabs, Ipswich, MA).
Strains Genotypes References or sources DNA for sequencing, ligation, and random primer labeling
Ustilago maydis reactions was purified using the QIAquick gel extraction kit
FB2 a2b2 Banuett & Herskowitz (Qiagen, Valencia, CA).
(1989)
FB1 a1b1 Banuett & Herskowitz
(1989)
PCR conditions
LV20 a2b2 Dpao<CbxR This study
LV39 a1b1 Dpao<CbxR This study Routine PCR was conducted using Taq DNA polymerase
LV24(5) a2b2 Dodc/pao<HygR/ This study (Invitrogen) and the following general program: an initial
CbxR
cycle of 94 1C for 5 min, amplification (30–35 cycles) at 94 1C
LV24(7) a2b2 Dodc/pao<HygR/ This study
for 30 s followed by annealing at primer-specific temper-
CbxR
LV201 a2b2 Dpao<CbxR/ This study ature for 60 s, and polymerization at 72 1C (1 min kb1
pLV28 of DNA target length). When required, the expanded
LG4 a2b2 Dodc<HygR Guevara-Olvera et al. (1997) high fidelity PCR system (Boehringer, Mannheim, Ger-
LV54 a2b1 Dspe-sdh<CbxR Valdés-Santiago et al. (2009) many) was used according to the manufacturer’s instruc-
LV38C a1b1 Dodc/pao<HygR/ This study tions. An extension period of 7 min at 72 1C was
CbxR
programmed for those PCR products that were cloned into
Escherichia coli
pCR2.1 Topo (Invitrogen). The primers used are described
Top 10 F0 Invitrogen
in Table 2.

FEMS Yeast Res 10 (2010) 928–940 

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
930
Table 2. Primers used in this study
Primers Sequences
UEN13 5 0 -TGAACGAGAGCGACGAGGCTG-3 0
UEN14 5 0 -CATGGCGCACGACAGACAG-3 0
UEN15 5 0 -CTGACCGACATGCACGCACAC-3 0
UEN16 5 0 -CTTCGCAATCGACGCATTCG-3 0
UEN17 5 0 -GGCGCACTTGGGCAACATG-3 0
UEN9 5 0 -CCTCTTGCTGTCTCTGCCTTG-3 0
UEN10 5 0 -ATCTTCGCTCAACTCGTCTCG-3 0
PaoFow 5 0 -ACGTCCCCATATCCGGCC-3 0
5 0 -odc 5 0 -CAACATGGACGAGCTCGAAAAGAT-3 0
3 0 -odc 5 0 -GTAAGCGCCCATGTTTTCGTAGAC-3 0
LVPPaoFow 5 0 -CAGCGCCAGCAACACAAC-3 0
Genetic transformation of U. maydis
We used a slightly modified version of the procedure
described by Tsukuda et al. (1988). Protoplasts [50 mL,
obtained according to Wang et al. (1988)] were mixed with
1 mg of transforming DNA and 1 mL heparin [15 mg mL1 in
STC (10 mM Tris-HCl pH 7.5, 100 mM CaCl2, 1 M sorbi-
tol)], and incubated on ice for 10 min. At this time, 0.5 mL
ice-cold 40% polyethylene glycol (PEG MW 3350) was
added, and incubation continued for 15 min on ice. STC
(0.5 mL) was added, and the material was mixed by inver-
sion of the tube. Protoplasts were centrifuged at 1000 g
for 5 min, suspended in STC, and spread over double-
strength selective medium agar plates containing 1 M
sorbitol. After 4–5 days, colonies were transferred to fresh
plates containing the selective agent to confirm their
phenotype.
Transformation of U. maydis by electroporation was
carried out by a method described in http://www.upstate.
edu/biochem/amberg/protocols/yeast_genomic_DNA.php
(D. Amberg, pers. commun.).
DNA sequencing and analysis
DNA sequencing was performed using double-stranded
templates in an ABI PRISM 377 DNA automatic sequencer
(Perkin Elmer, Cambridge, UK). Homology searches were
performed using the FASTA3 searches of EMBL, SWISS-
PROT, and SWALL databases. Analysis of nucleotide se-
quences was done with the DNASTAR, DNASTRIDER 1.1, and
TRANSFAC programs.
Construction of plasmids for the isolation and
disruption of the gene-encoding polyamine
oxidase (PAO)
The sequence of gene PAO was obtained from the U. maydis
genome sequence at http://mips.gsf.de/genre/proj/ustilago/
(UM05850). The complete gene plus 1100 and 944 bp,
corresponding to the 5 0 - and 3 0 -noncoding regions, respec-

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
L. Valdés-Santiago et al.
tively, was amplified with primers UEN16 and UEN15 (see
Orientations Table 2), and the PCR product was cloned into pGEMT-easy
to produce plasmid, pAC2108. This plasmid was digested
Forward
Reverse with HindIII, which removed most of the ORF, substituted
Reverse by the carboxin-resistance cassette obtained by HindIII
Forward digestion from pCBX-AC2 (Valdés-Santiago et al., 2009) to
Forward generate pLV24 (pao<CbxR).
Forward
Reverse
Construction of a plasmid to transform pao
Forward
Forward
mutants with the wild-type PAO allele
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Reverse PAO gene was recovered from pAC2108 by digestion with
Forward
BamHI/NotI and ligated into the same sites of the selectable
autonomous replicating plasmid pHyg101 (Mayorga &
Gold, 1998) to generate the pLV28 plasmid. This was used
to complement pao mutants by electroporation.
Mating and virulence assays
Mating was analyzed by the Fuz reaction (Banuett, 1992,
1995). Virulence assays followed essentially the method
described by Martı́nez-Espinoza et al. (1997). Disease symp-
toms (chlorosis, anthocyanin formation, and tumor forma-
tion) were recorded routinely after 5–15 days unless
otherwise specified.
Isolation of segregants from inoculated
maize plants
The procedure described by Chavez-Ontiveros et al. (2000)
was followed. Mature tumors were excised from plants
infected with sexually compatible strains, and surface sterilized
with a commercial solution of 7% sodium hypochlorite for
5 min, followed by 70% ethanol for the same time, and
washed twice with sterile distilled water. Tumors were crushed
to liberate teliospores, which were treated with 0.5% CuSO4
for 1 h, filtered through cheesecloth, and recovered by centri-
fugation. Teliospores were washed with sterile distilled water,
diluted to a density of 104 mL, and aliquots of 0.1 mL were
spread onto plates of CM plus additions. After 24 h, telio-
spores had germinated to produce sporidia. These were
recovered, appropriately diluted, and inoculated onto fresh
plates. Smooth colonies were recovered for mating-type
determination with tester strains and for phenotypic analysis.
Stress assays
Different stress conditions were tested on solid media. Cells
were grown in liquid MM without polyamines for two cycles
to deplete the cell polyamine pools (Guevara-Olvera et al.,
1997). They were then collected by centrifugation and
washed with sterile distilled water by centrifugation. Cell
suspensions were adjusted to contain 108 cells mL1
(counted with a Neubauer chamber), 10-fold serial dilutions
were prepared, and 10 mL of each dilution was spotted on
FEMS Yeast Res 10 (2010) 928–940
Life without putrescine
plates containing different additions. In a different type of
hydrogen peroxide (H2O2) sensitivity assay, U. maydis
strains were plated on CM. Filter disks (eight per strain) of
Whatman paper (5 mm in diameter) were soaked with 10 mL
H2O2 (3% v/v) and placed over the plates. Plates were
incubated at 28 1C for 48 h, photographed, and the diameter
of the inhibition zones was measured.
Determination of growth rate
To monitor rate of cell growth, we incubated 106 cells mL1 in
triplicate samples of 15 mL of MM at 28 1C under shaking
conditions, and OD600 nm was measured at 2-h intervals for 24 h.
Polyamine analyses
Polyamines were extracted from the cells with 6% perchloric
acid for 3 h at room temperature. Derivatization of poly-
amines and analysis were done as described previously
(Valdés-Santiago et al., 2009), except that a gradient of
methanol in distilled water of 40–65% was used to elute the
polyamines from the HPLC column.
Microscopic observations
Microscopic observations were performed with a Leica
DMRE microscope. Photographs were obtained with a Spot
digital camera (Diagnostic Instruments, Livingston, UK).
Results
Identification and isolation of U. maydis
PAO gene
A comparative analysis of the proteins involved in the
catabolism of polyamines shows a high conservation be-
tween eukaryotes. We therefore used the human polyamine
oxidase protein sequence to identify a polyamine oxidase in
U. maydis, and its encoding gene. BLAST homology searching
of the National Center for Biotechnology Information
genome database revealed a single hypothetical homolog
gene in the U. maydis genome: UM05850. This was located
in chromosome 20, contig 214: 152339–1540721. The
U. maydis gene (1734 bp) encodes a protein (GenBank
accession no. FN689796) made of 577 amino acids. The
protein sequence contains the conserved FAD-binding motif
(GAGWSG) at the N-terminal region characteristic of poly-
amine oxidases, as well as the peroxisomal targeting signal
sequence, –SRL, at the C terminus (not shown). The
inspection of the U. maydis Pao sequence also indicated, in
common with polyamine oxidases from other organisms,
the presence of the consensus sequence (S/A/C/P)-(K/H/R)-
(I/L/M) (Gould et al., 1989), which is not cleaved after
protein import into the peroxisome, where its oxidation
product (H2O2) is degraded by catalase (Wu et al., 2003).
FEMS Yeast Res 10 (2010) 928–940
931
To isolate the gene, a DNA fragment (3775 bp in length)
containing the PAO gene ORF (1731 bp) plus 1100 bp
upstream and 944 bp downstream of the ORF was amplified
by PCR using specific primers UEN16 and UEN15 (see Table
1), and cloned into pGEMT, to obtain pAC2108 (see
Materials and methods).
Disruption of the gene-encoding U. maydis
polyamine oxidase (PAO)
To determine the specific role of putrescine in U. maydis, we
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proceeded to disrupt the gene-encoding polyamine oxidase
using as background an odc mutant (LG4) that is unable to
produce putrescine from ornithine (Guevara-Olvera et al.,
1997). Primers UEN16 and UEN15 were used to amplify the
cassette containing the carboxin-resistance gene flanked by
the 5 0 - and 3 0 -untranslated regions corresponding to the
promoter and terminator of PAO gene from pLV24 plasmid
(see Materials and methods) and used to transform proto-
plasts of the LG4 (odc<HygR) mutant. Transformants were
recovered on plates of a medium containing hygromycin,
carboxin, putrescine, and spermidine, and were probed by
PCR using primers PaoFow and UEN14, the first one
located 840 bp upstream from the disruption cassette, and
the second one located within the cassette. A band of
4619 bp indicated the presence of the mutant allele at the
right locus, while a 3806 bp band corresponded to the wild-
type gene. The putative transformants were verified using
primers PaoFow and UEN9 (whose sequence is within the
carboxin-resistance gene). Amplification of a 3761-bp frag-
ment confirmed the nature of the double mutants. From the
transformants obtained, we selected two odc/pao mutants,
denominated, LV24(5) and LV24(7) for further studies.
These were further characterized by a Southern blot assay
after digestion with SacI, using an 800-bp fragment belong-
ing to the PAO ORF as a probe. The absence of bands in both
mutants indicated the absence of the gene (Fig. 1). Double
mutants of different mating genotypes were isolated by
genetic recombination in planta with strain FB1 (a1b1), as
described in Materials and methods, and an a1b1 double
mutant (LV38C) was selected for further studies. As ex-
pected, odc/pao mutants were resistant to carboxin and
hygromycin, and auxotrophic to putrescine or spermidine
(Fig. 2). This last result is evidence that putrescine serves as
an intermediate in spermidine biosynthesis, and is not
required for vegetative growth of U. maydis.
Determination of polyamines in
odc/pao mutants
To confirm that the double mutants LV24(5) and LV24(7) were
unable to synthesize putrescine, we proceeded to measure their
polyamine content by HPLC. As control we analyzed the
parental LG4 strain. No putrescine was detected in odc/pao

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
932 L. Valdés-Santiago et al.

(a)

cI
cI

cI

Sa
Sa

Sa
3188 bp 2783 bp

Hi III
III

Hi III
Hi dIII
III
nd
nd

nd

nd
n
Hi

Hi
PAO ORF
PAO Promoter Probe
cI

cI
Sa

Sa
894 bp

Fig. 1. Confirmation of odc/pao mutants.

Carboxine resistance

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gene (a) Schematic representation of the PAO gene,
and the disruption cassette. (b) PCR analysis to
(b) (c)
identify the presence of PAO gene using primers
PaoFow and UEN14. A 4619-bp fragment
4619 bp corresponds to the mutant gene, and a 3806-bp
fragment to the wild-type gene. (c) Southern blot
3806 bp 3188 bp analysis of the parental LG4 strain and LV24(7)
2738 bp odc/pao mutant digested with SacI, and probed
with a fragment from PAO ORF obtained by
1kb LV24(5) LV24(7) LG4 LV24(7) LG4 digestion with HindIII.

FB2

LV24(5) LV24(7)

LG4

MM7 Spd (0.1 mM) Put (5 mM)

Fig. 2. Auxotrophic phenotype of odc/pao mutants. Wild type (FB2), parental (LG4) and odc/pao mutants [LV24(5) and LV24(7)] were inoculated on
plates of solid MM containing the indicated additions, incubated for 48 h at 28 1C and photographed. Spd, spermidine; Put, putrescine.

double mutants, contrary to what occurred in the parental
strain (Fig. 3). It is notable that this is the first report of
eukaryotic mutants that contain spermidine only, and consti-
tutes evidence that putrescine is not essential for cell growth, at
least in the case of U. maydis, and probably other fungi.
Isolation of pao single mutants
By use of genetic recombination in planta between the
double mutant LV24(7) (odc/pao a2b2) and the wild-type
strain FB1 (a1b1) (see Materials and methods), we were able
to isolate the corresponding recombinants. Teliospores were
germinated on plates of CM containing carboxin and
spermidine, and the meiotic products were transferred to
fresh plates to test their growth on media containing
spermidine and either carboxin or hygromycin. Recombi-
nants were further selected, and their mating type and
auxotrophy to spermidine were determined. Several
mutants were thus confirmed by PCR using primers PaoFow
and UEN14. The presence of the wild-type ODC gene was
further confirmed by PCR using primers 5 0 -odc and 3 0 -odc
(we expected an 857-bp fragment for the wild-type gene,
and a 2700-bp fragment for the mutant gene). Using this
procedure we were able to isolate single pao mutants of
different mating genotypes (Table 1 and Fig. 4).
Phenotypic analysis of double and single pao
mutants
Growth rate
pao mutants showed only a slightly reduced growth rate at
28 1C in both MM and CM containing 0.1 mM spermidine
as compared with the wild-type strain, whereas a higher


c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 928–940
Published by Blackwell Publishing Ltd. All rights reserved
Life without putrescine 933

2000 100 100

1 (a) 150 (b)
90 90
1750

% Methanol
80 80

% Methanol
1500 100 2
70 70
1250 60 60
150
1000 50 50
mAU

mAU
40 100 40
750
2 30 1 30
500
20 50 20
250 10 10
0
0 0

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8 9 1 1 1 1 1 1 1 1 1 1 2 8 9 1 1 1 1 1 1 1 1 1 1 2
0 1 2 3 4 5 6 7 8 9 0 0 1 2 3 4 5 6 7 8 9 0
Time (min) Time (min)

100
(c)
60 90
80

% Methanol
70
40 60
2 50
mAU

40
20 30
20
10

0 8 9 1 1 1 1 1 1 1 1 1 1 2
0 1 2 3 4 5 6 7 8 9 0
Time (min)

Fig. 3. Chromatographic separation of the benzoylated derivatives of free polyamines. (a) Elution profile of a mixture of putrescine and spermidine
standards (1 and 2, respectively). (b) Analysis of polyamines in the parental strain LG4. (c) Polyamine determination in LV24(7) odc/pao mutant. Data are
representative of four different experiments. Elution times and profiles were taken directly from the recorder data (the arrows indicate the peak
corresponding to putrescine).

(a) (b)

2700 bp
Fig. 4. Confirmation of single pao mutants.
(a) PCR analysis to identify the PAO and pao
alleles using PaoFow and UEN14 primers.
4619 bp
Fragments of 3806 and 4619 bp correspond to
850 bp
the wild-type and mutant genes, respectively. For
3806 bp
comparison, the double odc/pao mutant and its
odc parental strain (LG4) are included. (b) PCR
analysis to identify the presence of the wild-type
ODC gene in a single pao mutant using primers
5 0 -odc and 3 0 -odc. For comparison the double
odc/pao mutant and its odc parental strain (LG4)
are included. A fragment of 850 bp corresponds
to the wild-type gene, while a 2700-bp fragment
corresponds to the mutant allele.

reduction in growth was observed in double odc/pao byproduct of Pao catalysis (N-acetyl-3-aminopropanalde-
mutants than in pao single mutants in the same media hyde) has been described as a precursor of b-alanine (a
(results not shown). It is important to remember that the component of pantothenic acid) in Saccharomyces cerevisiae

FEMS Yeast Res 10 (2010) 928–940 

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
934
(White et al., 2001), but in most of the studied eukaryotes,
including fungi, b-alanine is synthesized by b-alanine dehy-
drogenase as a degradation product of uracil (e.g. Gojkovic
et al., 2001). Our search in the U. maydis genome (http://mips.
helmholtz-muenchen.de/genre/proj/ustilago) also revealed the
presence of a homolog gene-encoding b-alanine dehydrogen-
ase in this fungus, suggesting that this, and not the pathway
from spermidine, is the important source of b-alanine. This
hypothesis is supported by the observation that pao mutants
grow in a MM without any auxotrophic requirement.
Dimorphic transition
Wild-type haploid cells of U. maydis grow at pH 3 in the form
of mycelium, and at pH 7 they display a yeast-like phenotype
(Ruiz-Herrera et al., 1995). However, odc mutants require a
high concentration of putrescine to undergo this morpholo-
gical transition (Guevara-Olvera et al., 1997). When we
induced the yeast-to-hypha transition by changing the pH in
the LV20 (pao) mutant, we did not observe any difference
with respect to the wild-type strain. In the case of LV24(7)
(odc/pao) mutant, its behavior was the same as odc single
mutants, and its mycelial growth occurred only in the presence
of spermidine or high putrescine concentrations (Fig. 5).
Response to stress
When mutants LV24(7) (odc/pao) and LV20 (pao) were
subjected to ionic or osmotic stress (1 M NaCl, 10 mM LiCl,
1 2
(a)
(b)
(c)
Fig. 5. Cellular morphology of wild-type, odc/pao, odc, and pao strains. Cells were grown in MM at pH 3, containing, respectively: (a) 0.5 mM
putrescine; (b) 5 mM putrescine; (c) 5 mM spermidine. 1, FB2; 2, LV20 pao; 3, LG4 odc; 4, LV24(7) odc/pao.

c 2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
L. Valdés-Santiago et al.
or 1 M sorbitol), only the double mutant displayed a
sensitive phenotype to these conditions, whereas the simple
LV20 pao mutants showed no differences to the wild-type
strain (Fig. 6). The response to oxidative stress was different.
In the agar diffusion test, the halos of inhibition shown by
LV20 and LG4 (pao and odc mutants, respectively) were
wider in comparison with the wild-type strain (Fig. 7a and
b), whereas, interestingly, the halo from the LV24(7) (odc/
pao) mutant was narrower than the one from the parental
strain. The same relative results were obtained when inhibi-
tion was measured on solid medium containing H2O2 and
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inoculated with decimal dilutions of the cell suspensions
(Fig. 7c).
Determination of mating
Fuz reaction of sexually compatible odc/pao double mutants
occurred at all the concentrations of spermidine used, but
required high levels of putrescine (5 mM). In contrast,
mating of compatible pao mutants was not affected by the
absence of spermidine (not shown).
Virulence of mutants
Maize seedlings inoculated with mixtures of sexually com-
patible odc/pao mutants did not develop tumors, in contrast
to plants inoculated with wild-type cells. On the other hand,
inoculation of maize seedlings with compatible mixtures of
pao mutants induced tumors, but these were noticeably
3 4
FEMS Yeast Res 10 (2010) 928–940
Life without putrescine 935

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
106

105

104

103
(a) (b) (c) (d)

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Fig. 6. Effect of different stress conditions on the growth of Ustilago maydis. Cells were grown for 24 h in MM containing 0.1 mM spermidine followed
by two growth cycles in the absence of spermidine to deplete polyamine pools. Cell suspensions were adjusted to a density of 108 cells mL1, decimally
diluted, and inoculated on solid MM containing 0.1 mM spermidine and (a) nothing (control), (b) 1 M NaCl, (c) 1 M sorbitol, or (d) 10 mM LiCl. 1, Wild-
type strain FB2; 2, single odc mutant LG4; 3, double odc/pao mutant LV24(7); 4, single LV20 pao mutant. Plates were photographed after 48 h of
incubation at 28 1C.

(a)

FB2 LG4 (odc) LV24(7) LV20 (pao)

(odc /pao)

(b) 2.5

2
Halo size (cm)

1.5

0.5

0
FB2 LG4 (odc) LV24(7) LV20 (pao)
(odc /pao)

(c) (d)
)
ao
c /p

)
od

O
LV o)

)
/p )

ao

ao

ao
)

)
dc (7

PA

PA
)(
dc

dc

ao 1

ao 1
a

ao
(o 24

(p V20

(p V20
(p

(p

(p

(p
(7
(o

(o

/p

/p
20

20

24

20

20
4

4
2

2
L

L
LG

LG
FB

FB

FB

FB
LV

LV

LV

LV

LV

106

105

104

Fig. 7. Sensitivity of Ustilago maydis wild-type strain and mutants to oxidative stress. (a) Sensitivity of strains to H2O2 assessed in an agar diffusion test in
which filters soaked with H2O2 (0.8 mM) were placed over plates of solid MM containing 0.1 mM spermidine, previously inoculated with the indicated
strains. At the end of 48 h, the plates were photographed. (b) Diameter of the halos quantified for the strains given in (a). Error bars indicate SDs derived
from three independent experiments consisting of eight replicas each. (c) Serial 10-fold dilutions of cell suspensions were spotted on solid MM plates
supplemented with 0.1 mM spermidine only (left) or spermidine plus 0.8 mM H2O2 (right). (d) Similar spot test using a complemented strain of LV20
mutant with pLV28 (LV201). Left, control plate; right, plate containing 0.8 mM H2O2.

FEMS Yeast Res 10 (2010) 928–940 

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
936 L. Valdés-Santiago et al.

smaller, and appeared in a smaller number of plants as Discussion

compared with the wild-type strains. Moreover, infected
plants appeared healthy at a time when those infected with
wild-type strains looked dwarfed or dead (Fig. 8 and Table 3).
Complementation of pao mutants
Plasmid pLV28 carrying the PAO gene was introduced into
the LV20 mutant (a2b2 pao<CbxR), and transformants were
recovered, and confirmed by their resistance to hygromycin,
In different systems, evidence has been obtained suggesting
that spermidine, contrary to putrescine, is absolutely neces-
sary for eukaryotic cell growth (Balasundaram et al., 1991;
Chattopadhyay et al., 2003; Valdés-Santiago et al., 2009).
Nevertheless, whether putrescine serves only as a spermidine
precursor or also has other functions in cellular metabolism,
has been a matter of discussion, mainly because in the
studied systems it is difficult to isolate its effects from those
of the other polyamines present in the cell. Several authors

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and by PCR using UEN13 and UEN15 and LVPPaoFow and
UEN15 primers, which amplified a band of 800 bp corre-
sponding to the terminator area, and the whole PAO gene,
respectively (results not shown). We observed that transfor-
mants recovered resistance to H2O2 to a higher level than the
wild-type strain (see Fig. 7d), possibly because of their
higher number of PAO copies in the autonomous replication
plasmid. In the same way, when a transformant (LV201) was
inoculated into maize plants together with a pao mutant
LV39 (a1b1 pao<CbxR), they were shown to be as virulent as
the wild-type strains (Fig. 8e and Table 3).
have suggested that putrescine itself might have important
physiological effects (Emanuelsson & Heby, 1978; Pegg,
1988), and in several plants putrescine catabolites have been
found to serve as precursors of important alkaloids (Cohen,
1998; Hartmann, 1999). It is also known that, in vitro,
putrescine stimulates both the cleavage reaction and the
catalytic activity of the mature S-adenosylmethionine
(SAM) decarboxylase (Stanley & Pegg, 1991). On the other
hand, Yuan et al. (2000) demonstrated that putrescine by
itself was not essential for growth and migration of intestinal
epithelial crypt cells in vitro.

(a) (b) (c) (d)

(e)
A B
A B

Fig. 8. Infection symptoms of maize seedlings inoculated with mixtures of different sexually compatible Ustilago maydis strains. (a) (A) plants inoculated
with FB2 (a2b2 wild-type)  FB1 (a1b1 wild-type) or (B) plants inoculated with LV24(7) (a2b2, odc/pao)  LV38C (a1b1, odc/pao). (b) (A) plants
inoculated with FB2 (a2b2 wild-type)  FB1 (a1b1 wild-type) or (B) plants inoculated with LV20 (a2b2, pao)  LV39 (a1b1, pao). White arrows point to a
small tumor in the leaf, and one exceptionally large tumor at the base of a plant. Notice the general appearance of the plants inoculated with wild-type
or pao strains. (c and d) Magnification to show the tumors in plants of (b) (white arrows). (e) Plants inoculated with LV201 (a2b2, pao/pPAO)  LV39
(a1b1, pao).

Table 3. Virulence of mutants compared with wild-type strain

Strains No. of plants Total no. of plants Plants with tumors 4 5 mm Plants with tumors o 5 mm
FB1  FB2 79 60/79 (75.9%) 9/79 (11.4%) 51/79 (64.5%)
LV24(7)  LV38C 20 0 0 0
LV39  LV20 143 81/143 (56.6%) 28/143 (19.6%) 53/143 (37%)
LV39  LV201 40 35/40 (87.5%) 5/40 (12.5%) 30/40 (75%)


c 2010 Federation of European Microbiological Societies FEMS Yeast Res 10 (2010) 928–940
Published by Blackwell Publishing Ltd. All rights reserved
Life without putrescine
An additional source of difficulties in determining speci-
fic roles for putrescine is that it is synthesized by two
mechanisms: (1) directly by decarboxylation of ornithine
by Odc and (2) by the catabolism of spermine and spermi-
dine. Polyamine catabolism is catalyzed by two enzymes:
spermidine/spermine N1-acetyltransferase and the FAD-
dependent polyamine oxidase (Pao). Pao catalyzes the
oxidative splitting of acetyl derivatives of spermidine or
spermine to form putrescine or spermidine, respectively
(Seiler, 1995; Wang et al., 2001). There exists evidence
suggesting that this retroconversion is involved in the
regulation of intracellular polyamine levels, and also in
different cellular processes such as apoptosis. In plants it
contributes to the hypersensitivity response leading to
induced cell death (Cohen, 1998; Yoda et al., 2003). Accord-
ingly, to obtain a decisive answer to the question as to
whether putrescine has some specific roles other than merely
to serve as a spermidine precursor, it was necessary to obtain
a system completely free of intracellular putrescine, by
inhibition of the two pathways leading to its biosynthesis.
In this study, we took advantage of the fact that U. maydis
does not contain spermine, and proceeded to isolate mutants
devoid of Odc and Pao (odc/pao double mutants), which did
not contain putrescine as demonstrated by HPLC analysis,
and analyzed their phenotype. These double mutants behaved
as putrescine or spermidine auxotrophs, the same as the single
odc mutants, showing that putrescine is not necessary for
vegetative growth, and that spermidine is the most important
polyamine in this respect. This result of course does not
invalidate the role of putrescine in the activation of SAM
decarboxylase, a function that was not possible to analyze in
our odc/pao double mutants, which absolutely require the
addition of putrescine or spermidine to grow.
Another phenomenon where putrescine appears not to be
directly involved is the dimorphic transition of U. maydis, as
odc/pao double mutants behave like the single odc mutant
(LG4; Guevara-Olvera et al., 1997) in their polyamine require-
ments for dimorphic transition under acid conditions.
Regarding virulence, we have reported that U. maydis spe
mutants showed only attenuated virulence to maize,
whereas odc mutants were completely avirulent (Valdés-
Santiago et al., 2009), similar to the odc/pao double mutants.
This difference suggests that mutants are able to obtain a low
supply of spermidine but not putrescine from the plants
(maize plants contain higher amounts of spermidine and
spermine than putrescine; Rodrı́guez-Kessler et al., 2008).
The most plausible explanation for this difference in viru-
lence is that putrescine is somehow required, at least
partially, for U. maydis virulence, possibly due to its role in
protection to stress (see Results). Considering the back-
ground of the odc/pao double mutants, their avirulence was
anticipated (Valdés-Santiago et al., 2009), but the reduced
virulence of pao mutants was unexpected. It is possible that
FEMS Yeast Res 10 (2010) 928–940
937
the defect in virulence of pao mutants may be related to their
increased sensitivity to oxidative stress. It is known that the
oxidative killing of pathogenic fungal cells by the host
defense mechanisms represents an important line of elim-
ination of pathogenic microorganisms (Montesano et al.,
2003; Nürnberger et al., 2004). Ustilago maydis mutants
affected in Yap, a transcription factor involved in oxidative
response showed increased sensitivity to H2O2, and their
virulence was attenuated (Molina & Kahmann, 2007). An
alternative possibility is that these mutants have an altered
polyamine homeostasis, and become more sensitive to the
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stress imposed by the host.
One peculiar observation was the increased resistance of
odc/pao mutants to H2O2 as compared with single odc or pao
mutants, an odd result difficult to explain. A possible
explanation would be that, in vitro, spermidine was more
efficient than putrescine in providing protection to oxida-
tive stress, indicating a competition between the poly-
amines. As only the double mutant lacks putrescine, in a
medium with an excess of spermidine it would be more
resistant to H2O2 than would either single mutant.
It is important to recall that no general agreement exists
on the role of specific polyamines in the protection against
oxidative stress; for example, putrescine protected E. coli
single samdc and double odc/samdc mutants against oxida-
tive stress (Chattopadhyay et al., 2003), but on the other
hand, spermine caused a reversion of an apoptotic pheno-
type induced by oxidative stress in spe/pao double mutants
of S. cerevisiae (Chattopadhyay et al., 2006). Similarly, in
plants, putrescine, Odc, and arginine decarboxylase levels
increased in response to oxidative stress (Szigeti et al., 1996;
Ye et al., 1997), but Trypanosoma cruzi lipoperoxidation
induced by H2O2 was avoided by spermine, and putrescine
had no effect at all (Hernández et al., 2006).
The observation that U. maydis odc/pao double mutants
showed increased sensitivity to osmotic and ionic stress as
compared with odc mutants, even when growing in the
presence of spermidine, suggests that putrescine is involved
in the resistance of the fungus to these types of stress. This
hypothesis agrees with the observation that under salt stress,
different cereals showed increased levels of putrescine and
decreased levels of spermidine (Simon-Sarkadi et al., 2002).
Likewise, putrescine content in barley, corn, wheat, and oat
was increased up to 60-fold when plants were exposed to
osmotic stress (Flores & Galston, 1982).
Systems where polyamine catabolism has been affected
show only slight changes in polyamine homeostasis (e.g.
Niiranen et al., 2006), suggesting the existence of other
regulatory mechanisms for polyamine accumulation, possi-
bly at the levels of biosynthesis or excretion (Casero & Pegg,
1993; Seiler, 2004). These data disagree with the phenotype
of U. maydis pao mutants, which showed a modest but
significant reduction in growth rate with increased

c2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
938
sensitivity to oxidative stress, and reduced virulence. These
results indicate that if indeed polyamine oxidase is not of
vital importance to the cell, it is still important for the
ecological success of U. maydis in its natural environment.
From the results obtained in this study, we may conclude
that putrescine is not necessary for the growth of U. maydis, its
main role being to serve as an intermediary in the biosynthesis
of spermidine, which is absolutely required by the fungus. In
addition, putrescine is not required for the dimorphic transi-
tion, but appears to be involved in the protection against some
forms of stress and, interestingly, in virulence. Evidence was
also provided that the retroconversion of spermidine into
putrescine may not be very important under laboratory
conditions, but taking into consideration some aspects of the
phenotype of pao mutants, it appears to be important for the
success of the fungus in nature.
Acknowledgements
The present work was partially supported by Consejo
Nacional de Ciencia y Tecnologı́a (CONACYT), Mexico,
and Consejo de Ciencia y Tecnologı́a del Estado de Guana-
juato (CONCYTEG), Mexico. We are grateful to Shilpa
Wagh, Claudia León, Lucila Ortı́z, Yesenia Ruiz, and Alicia
Mireles for their invaluable help in technical assistance.
L.V.S. is a doctoral student supported by a fellowship from
CONACYT, and J.R.-H. is Emeritus National Investigator
from Sistema Nacional de Investigadores, Mexico.
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