Fungal Genetics and Biology 49 (2012) 48–57

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Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

Identification and functional characterization of indole-3-acetamide-mediated

IAA biosynthesis in plant-associated Fusarium species
Elena Tsavkelova a,1, Birgitt Oeser b, Liat Oren-Young a, Maayan Israeli a, Yehezkel Sasson c,
Bettina Tudzynski b, Amir Sharon a,⇑
a
Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel
b
Molekularbiologie und Biotechnologie der Pilze, Institut für Biologie und Biotechnologie der Pflanzen, Hindenburgplaz 55, 48143 Münster, Germany
c
Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv 69978, Israel

a r t i c l e i n f o a b s t r a c t

Article history: The plant hormone indole-3-acetic acid (IAA) can be synthesized from tryptophan via the intermediate
Received 25 June 2011 indole-3-acetamide (IAM). The two genes, IaaM (encoding tryptophan monooxygenase) and IaaH (encod-
Accepted 24 October 2011 ing indole-3-acetamide hydrolase) that constitute the IAM pathway have been described in plant-asso-
Available online 4 November 2011
ciated bacteria. We have identified putative homologs of the bacterial IaaM and IaaH genes in four
Fusarium species – Fusarium proliferatum, Fusarium verticillioides, Fusarium fujikuroi, and Fusarium oxyspo-
Keywords: rum. In all four species the two genes are organized next to each other in a head to head orientation and
Auxin
are separated by a short non-coding region. However, the pathway is fully functional only in the orchid
IAA
IAM
endophytic strain F. proliferatum ET1, which produces significant amounts of IAM and IAA. Minor
Fungi amounts of IAM are produced by the corn pathogen F. verticillioides strain 149, while in the two other spe-
Fusarium cies, the rice pathogen F. fujikuroi strain m567 and the tomato pathogen F. oxysporum f. sp. lycopersici
IaaM strain 42-87 the IAM pathway is inactive. Deletion of the entire gene locus in F. proliferatum ET1 resulted
IaaH in drastic reduction of IAA production. Conversely, transgenic strains of F. fujikuroi over-expressing the
Colletotrichum F. proliferatum IAM genes produced elevated levels of both IAM and IAA. Analysis of the intergenic
promoter region in F. proliferatum showed that transcriptional activation in direction of the IaaH gene
is about 3-fold stronger than in direction of the IaaM gene. The regulation of the IAM genes and the
limiting factors of IAA production via the IAM pathway are discussed.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Microorganisms, including bacteria and fungi, are also capa-

The plant hormone indole-3-acetic acid (IAA) is a major regula-
tor of plant growth and development. While the physiological role
of auxins is well understood, IAA biosynthesis in plants has only
partially been characterized. Tryptophan is considered the major
source of IAA synthesis in plants, but indole might also be used
in some cases (reviewed in Normanly, 2010). The most common
pathway involves three steps in which tryptophan is first deami-
nated to indole-3-pyruvic acid followed by decarboxylation of
IPA to indole-3-acetaldehyde and finally to IAA. Indole-3-acetalde-
hyde is a key intermediate in two additional pathways of IAA bio-
synthesis and can be generated directly from tryptophan or after
conversion of tryptophan to tryptamine (Zhao et al., 2002).
Abbreviations: IAA, indole-3-acetic acid; IAM, indole-3-acetamide; IPA, indole-
3-pyruvic acid; MP, mating population; GFC, Gibberella fujikuroi species complex;
HGT, horizontal gene transfer.
⇑ Corresponding author. Fax: +972 36405498.
E-mail address: amirsh@ex.tau.ac.il (A. Sharon).
1 been identified, ruling out that IAA can be generated through
Permanent address: Department of Microbiology, Biological Faculty, Moscow
State University, 1-12 Leninskie Gory, Moscow 119991, Russia.
ble of producing IAA (Tsavkelova et al., 2006; Tudzynski and
Sharon, 2001). Unlike the situation in plants, the physiological
role of microbial auxins is unclear, while the biosynthesis path-
ways have been analyzed in great details, and the correspond-
ing IAA biosynthesis genes were elucidated (Costacurta and
Vanderleyden, 1995; Patten and Glick, 1996). In addition to
the plant metabolic pathways, bacteria can also synthesize IAA
through indole-3-acetamide (IAM pathway). In this pathway
tryptophan is converted to IAM by tryptophan-2-monooxygen-
ase (encoded by IaaM) followed by further conversion of IAM
to IAA by the enzyme IAM hydrolase (encoded by IaaH). The
IAM pathway is mainly found in plant-associated bacteria, such
as Pseudomonas, Agrobacterium and Rhizobium species (Patten
and Glick, 1996). The corresponding genes, IaaM and IaaH, are
usually organized in a small operon and are highly conserved
among different bacterial species. Homologs of the bacterial
IaaH genes have been recently identified in plants (Mano
et al., 2010). However plant homologs of IaaM genes have not
this metabolic route in plants.

1087-1845/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2011.10.005
 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
IAA production in fungi is well documented and requires exter-
nal supply of tryptophan (Reineke et al., 2008; Tsavkelova et al.,
2006; Tudzynski and Sharon, 2001). Although tryptophan is used
as a precursor for IAA production in fungi, the main plant metabolic
route through indole pyruvic acid has been conclusively demon-
strated only in the smut fungus Ustilago maydis, the only fungal spe-
cies from which IAA metabolic genes have been isolated (Reineke
et al., 2008). Two indole-3-acetaldehyde dehydrogenase genes
(IAD1, IAD2) were identified in U. maydis, and single and double
mutants were generated through gene deletion. The Diad1/Diad2
double mutant strains were blocked in the conversion of both indole-
3-acetaldehyde and tryptamine to IAA. Furthermore, deletion of
two aromatic amino acid aminotransferases (TAM1 and TAM2,
required for conversion of tryptophan to IPA) in the Diad1/Diad2
mutant background resulted in a further decrease in IAA production.
It was previously shown that Colletotrichum gloeosporioides f. sp.
aeschynomene (C. gloeosporioides) produces large quantities of IAA
in axenic culture (Robinson et al., 1998). Unlike in other fungi,
the major IAA-biosynthesis pathway in C. gloeosporioides is the
bacterial IAM pathway, as indicated by accumulation of IAM in cul-
ture media, and detection of enzymatic activities of the IAM path-
way enzymes in fungal protein extracts (Robinson et al., 1998;
Maor et al., 2004). Despite intensive efforts, the corresponding
genes could not have been isolated by conventional genetic and
biochemical approaches.
The fast progress in sequencing technologies resulted in a
wealth of genomic information, and a large number of fungal gen-
omes are now publicly available (reviewed by Xu et al., 2006). We
searched the available fungal genomes using the bacterial IAM
pathway genes as references and identified a locus containing
putative homologs of the bacterial IaaM and IaaH genes in the dat-
abases of two Fusarium species: the corn pathogen Fusarium verti-
cillioides and the tomato pathogen Fusarium oxysporum (http://
www.broadinstitute.org/annotation/genome/fusarium_group/
MultiHome.html). Here we report on the isolation and functional
analysis of these genes in four Fusarium species.
2. Materials and methods
2.1. Fungal strains
IAA production was analyzed in the following Fusarium species:
Fusarium proliferatum ET1, mating population (MP)-D, previously
isolated from the roots of epiphytic tropical orchids (Tsavkelova
et al., 2003, 2008); Gibberella fujikuroi (MP)-C (anamorph Fusarium
fujikuroi) strain m567 (Fungal Culture Collection, Weimar, Germany),
a rice pathogen and a gibberellin-producing strain; F. verticillioides
strain 149 (MP-A), a corn pathogen (Oren et al., 2003); F. oxysporum
f. sp. lycopersici 42-87, a tomato vascular wilt pathogen (Di Pietro
and Roncero, 1998). All species were cultivated on potato dextrose
agar (PDA) and maintained at 25 °C in the dark. C. gloeosporioides
strain 3.1.3 was used as reference for IAA and IAM production.
For determination of indoles production, fungi were cultivated
in a 250 ml light-protected Erlenmeyer flask with 50 ml Czapek-
Dox (CD) medium (per liter: 3 g NaNO3, 0.5 g MgSO47H2O, 0.5 g
KCl, 55 mg FeSO4, 1.0 g KH2PO4, 30 g sucrose) supplemented with
either tryptophan or IAM. The flasks were incubated in darkness
at 28 °C on a rotary shaker with agitation at 180 rpm. Aliquots of
the culture media were sampled at various time points and indoles
were extracted and analyzed.
2.2. Indoles extraction and analysis
2.2.1. Total indoles
Relative indoles content was estimated by the Salkowski
method (Glickman and Dessaux, 1995; Gordon and Weber, 1951).
49
Samples were centrifuged at 7000g for 15 min, the supernatant
was collected and 1 ml of the supernatant was transferred into a
light protected tube and mixed with 1 ml of Salkowski reagent
(1.015 g FeCl36H2O, 150 ml H2SO4, 250 ml ddH2O). The pH was ad-
justed to 2.8 with 0.1 N HCl, the tubes were incubated for 15 min at
30 °C for color development and then absorbance at OD540 was
determined. Standard curves were prepared from serial dilutions
of 100 mM IAA stock solution. All experiments included three rep-
lications and were repeated at least three times.
2.2.2. TLC analysis
Fungal cultures were centrifuged, the supernatant was collected
and pH was adjusted to 2.8. Aliquots of 800 ll were extracted with
ethyl acetate (1:2, v/v) by vigorous shaking for 10 min. After phase
separation, the ethyl acetate fraction was removed to a light pro-
tected tube, the ethyl acetate was evaporated, and the solid residue
was dissolved in 30 ll of methanol. Samples were spotted onto a
silica gel 60 F254 plates (Merck), and developed with ethyl ace-
tate:isopropanol:ammonia solution (45:35:20) running solvent.
After development, the plates were dried, sprayed with van
Ehmann’s reagent [a 3:1 mixture of Salkowski’s and Ehrich’s
reagents (Ehmann, 1977)], and heated to 90 °C until the spots were
visualized. The Rfs and colors of the bands were compared with
1 mM each of IAA, IAM, TOL and Trp standard compounds.
2.2.3. HPLC analysis
HPLC was used for quantitative determination of IAA and IAM
levels. Following extraction with ethyl acetate the dry extract
was dissolved in 750 ll of 20% methanol: water (v/v) and filtered
through 0.22 lm filter disc. For HPLC analysis 10 ml samples were
injected to an HPLC instrument equipped with a guard column and
a C18 reverse phase Eclipse XDB column, 5 lm, 4.6  150 mm, and
a UV detector. Indoles were separated on 25–100% (v/v) MeOH:-
water gradient containing 2% acetic acid over 35 min with a flow
rate of 1 ml/min. Under these conditions, the retention times of
IAM and IAA were 6.5–7.2 and 15.8–17.6 min, respectively. Absor-
bance at OD280 was determined and integration of the different
peaks was calculated according to standard compounds. All exper-
iments included three replications and were repeated several
times.
2.3. Enzyme activity assay
Activity of the IAM pathway enzymes was determined by con-
version of Tryp and IAM to IAA according to Maor et al. (2004).
Fungi were cultured in CD medium for 3 days, the mycelium was
harvested, filtered through Miracloth and the biomass was imme-
diately grinded to a fine powder in liquid nitrogen. Mycelium pow-
der (5 g) was suspended in 11.8 ml of protein extraction buffer
[10 ml Tricine buffer (pH 8.3), 13.5 mM acetyl-trimethyl-ammo-
nium bromide (CTAB), and 200 ll of protease inhibitor cocktail
(Sigma–Aldrich)]. The tubes were incubated for 10 min at 28 °C
with shaking at 150 rpm, centrifuged at 7600g for 10 min and
the supernatant was divided into two separate tubes. Tryptophan
(5–10 mM) or IAM (2.5 mM) was added, and the tubes were incu-
bated at 28 °C in the dark with agitation at 100 rpm. Samples were
obtained at various times following incubations, extracted with
ethyl acetate and indoles were determined by TLC analysis.
2.4. DNA and RNA isolation and analyses
Plasmid DNA was extracted using Sigma GenElute Plasmid
Miniprep Kit (Sigma) according to the manufacturer’s protocol.
Fungal genomic DNA was isolated using standard procedures.
RNA was isolated from ground mycelium using a GenElute Mam-
malian Total RNA Miniprep kit (Sigma). For Northern blots, RNA
50 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
was isolated using an RNAgents total RNA isolation kit (Promega).
Northern blot analysis was performed according to Church and
Gilbert (1984) using 15 lg of total RNA. PCR analyses were per-
formed with 10–25 ng template DNA using standard programs.
RT-PCR was performed with the Reverse-iT 1st Strand Synthesis
Kit (ABgene). Total RNA was extracted, treated with DNase and
first-strand cDNA was synthesized from 1 lg. For semi-quantitative
analyses, cDNA samples were amplified by PCR, aliquots were re-
moved after 25, 30 and 35 cycles and analyzed by gel electropho-
resis. Sequences of oligonucleotides used in PCR and RT-PCR
reactions are presented in Suppl. Table S1. The primers for ampli-
fication of F. proliferatum ET1 and F. fujikuroi were designed accord-
ing to F. verticillioides and F. oxysporum published sequences
(http://www.broadinstitute.org).
2.5. Plasmids
Expression vectors were constructed for high constitutive
expression of each of the F. proliferatum ET1 IAM-pathway genes.
For expression of the putative tryptophan monooxygenase genes
(putative IaaM), a 1.87 kb genomic fragment containing the predicted
ORF was amplified from genomic DNA of strain F. proliferatum ET1
using primers F.prol-IaaM-BspHI-for and F.prol-IaaM-BamHI-rev
and cloned into pTZ57R plasmid (Fermentas). The fragment was
released by cutting with BspHI and BamHI restriction enzymes
and cloned into Ksh52-1 vector (Barhoom and Sharon, 2004) at
the BamHI and NcoI sites between the Aspergillus nidulans GPDA
promoter and TRPC terminator to produce the pOE-IaaM plasmid
vector. For expression of the IAM hydrolase gene (putative IaaH),
a 1.95 kb genomic fragment containing the predicted ORF was
amplified from genomic DNA of F. proliferatum ET1 using primers
IaaH-F1-Apa and IaaH-R1-Apa. The PCR product was digested with
ApaI restriction enzyme and cloned into pUCH2-8-nat vector,
which contains the nourseothricin resistance cassette from vector
pNR1 (Malonek et al., 2004). The promoter of F. fujikuroi GLNA gene
(Teichert et al., 2004) was amplified using primers glnA-F-HindIII
and glnA-R-SalI, and cloned into the HindIII/SalI-restricted vector
pUCH2-8-nat, carrying the IaaH gene from F. proliferatum ET1 to
create the IaaH over-expression vector pOE-IaaH.
For deletion of the entire putative IAM locus in F. proliferatum
ET1, the gene replacement vector pIAA-GR was generated by
sequentially cloning of the 0.7 kb 50 -and the 0.7 kb 30 -flanks of
the putative IAM locus in F. proliferatum ET1 into the vector
pNR1 such that the nourseothricin resistance cassette was flanked
by F. proliferatum genomic sequence. The flanks were amplified
using primers IAA-GR1-SacI/IAA-GR2-XbaI and IAA-GR3-HindIII/
IAA-SalI for the right and left flanks, respectively.
Two vectors were constructed with YFP and DsRED reporter
genes for assessment of the relative transcription activation of
the F. proliferatum ET1 non-coding region in the left (IaaH) and
right (IaaM) directions. The fragment was amplified twice: once
with primers that introduced NcoI and SacI restriction sites next
to the putative IaaH and IaaM genes, respectively, and then with
the same restriction sites but on the opposite sides of the fragment.
The PCR fragments were cloned into pYHN2 (Hoff and Kück, 2005)
next to the YFP coding sequence in place of the GPD promoter, pro-
ducing two plasmids: PIAY(+) and PIAY(). The dsRED coding se-
quence was amplified with primers introducing SacI and EcoRI at
the 50 and 30 ends of the gene, and cloned into the same sites in
the plasmids PIAY(+) and PIAY(). The resulting vectors were des-
ignated PIAYR(+) and PIAYR() (Fig. S1).
2.6. Genetic transformation
Genetic transformation of F. fujikuroi and F. proliferatum was
performed according to Malonek et al. (2005a) and Tudzynski
et al. (1996). Transgenic colonies were selected on medium with
hygromycin (Calbiochem) or nourseothricin (Werner BioAgents,
Jena, Germany), DNA was extracted and presence of the relevant
transgene was determined by PCR using the appropriate primers.
The following transgenic strains were produced: over-expression
of the putative F. proliferatum ET1 IaaH gene (plasmid pOE-IaaH, IAA
production was determined in three independent strains), over-
expression of both the putative IaaM and IaaH genes (co-transforma-
tion of plasmids pOE-IaaH and Ksh-IaaM, IAA production was
determined in three independent strains), deletion of both genes
(plasmid pIAA-GR, designated DDiaaM/iaaH, IAA production was
determined in three independent strains), expression of YFP and
dsRED from the endogenous IAM genes’ promoter [plasmids
PIAYR(+) and PIAYR()]. In all cases several isolates were produced,
a single insertion of the relevant plasmids was confirmed, and the
relevant phenotype was verified in a least three independent strains.
2.7. Microscopy and image analysis
Fluorescent microscopy was performed with a Zeiss Axioskp 2
epifluorescent microscope. YFP was visualized using Zeiss filter
set #46 (excitation 520/20 nm, emission 535/30 nm), dsRED was
visualized using Zeiss filter set #20 (excitation 546/12 nm, emis-
sion 575–640 nm). Images were captured using a Zeiss Exiocam
digital camera. For measurement of YFP and dsRED relative fluo-
rescence intensity, 50 spores of each strain were captured using
constant exposure parameters, number of pixels was determined
in each spore using the ImageJ 1.42q software (http://rsb.info.nih.-
gov/ij/), and the average fluorescence was calculated.
2.8. Calculation of specific activity of IAM genes’ bi-directional
promoter
Transgenic strains were produced, which express the F. prolifer-
atum intergenic region between the IaaM and IaaH genes fused on
each side to the reporter genes DsRED and YFP (see Fig. S1 for maps
of the vectors and results for details). Images were captured using a
fluorescent microscope with the Rhodamine and YFP Zeiss filter sets
#20 and #46, respectively. In addition to differences in fluorescence
intensity that result from the difference in expression activity of the
promoter in each direction, other parameters, which also affect fluo-
rescence intensity were taken into account: (i) the specific fluores-
cence of each, the YFP and DsRED proteins, (ii) variable expression
levels in different transgenic strains. Accordingly, the relative pro-
moter activity was calculated using the following set of equations:
I. Parameters and values
Specific effect of transgenic strain #1: x.
Specific effect of the transgenic strain #2: 1.
Specific fluorescence of YFP: a.
Specific fluorescence of dsRED: 1.
Promoter activity () direction: b.
Promoter activity (+) direction: 1.
dsRED YFP
Isolate 1 bx ax
Isolate 2 1 ba
II. Measurements
dsRED YFP
Isolate 1 N1 = 51 N2 = 22.19
Isolate 2 N3 = 6.5 N4 = 27.35
 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
III. Calculation
N2 a 22:19 N4 27:35
1: ¼ ¼ ¼ 0:435 ¼ ab ¼ ¼ 4:2
N1 b 51 N3 6:5
a To determine if the putative IAM gene clusters were functional
2: ¼ 0:435 ab ¼ 4:2 b ¼ 3:11 a ¼ 1:35
b
2.9. Sequence analyses
To determine the distribution of IaaH/IaaM and related genes,
blast searches were performed using bacterial IaaH and IaaM ami-
no acid sequences as query. Genomic blast analyses and pair wise
alignments were performed using the following databases:
http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi; http://ped-
ant.gsf.de/genomes.jsp?category=fungal; http://img.jgi.doe.gov/
cgi-bin/pub/main.cgi?section=FindGenes&page=findGenes; http:
//blast.ncbi.nlm.nih.gov/Blast.cgi.
Multiple alignments and phylogenetic analyses were performed
using Phylogeny.fr (http://www.phylogeny.fr/version2_cgi/in-
dex.cgi). Stop codons and frame shifts in the F. oxysporum IaaH
gene were ignored to produce an amino acid sequence suitable
for an alignment.
3. Results
3.1. Identification of the putative IAM gene cluster in fungal genomes
BLASTP search of available fungal genome databases was con-
ducted with the amino acid sequences of the bacterial tryptophan
monooxygenase (IaaM) and IAM hydrolase (IaaH) from Agrobacte-
rium tumefaciens (AJ237588.1, NP_059675.1) and Pseudomonas
syringae (AAR06971.2, NC_007005.1) (Fig. 1A). Sequences with
high homology to the bacterial IaaM gene were only found in F. ver-
ticillioides and F. oxysporum, whereas many putative amidases with
high homology to bacterial IaaH genes were identified in all fungal
genomes. A closer look revealed that in both F. verticillioides and F.
oxysporum there is a putative IAM hydrolase encoding gene next to
the putative tryptophan monooxygenase encoding gene. This is
similar to the situation in bacteria, in which the IaaM and IaaH
genes are placed next to each other in the same genetic locus
(Spaepen et al., 2007). The two genes are organized in a head to
head orientation and are separated by a 1854 bp non-coding region
in F. verticillioides (Fig. 1B). This putative IAM locus is missing in
Fusarium graminearum, but we were able to amplify a highly sim-
ilar region from two additional Fusarium species, F. proliferatum
ET1 and F. fujikuroi IMI58289, both belonging to the G. fujikuroi
species complex (GFC) (Leslie and Summerell, 2006). The genomic
regions containing the IAM gene clusters of F. proliferatum ET1 and
F. fujikuroi IMI58289 were cloned and sequenced (JN166965 and
JN166968, respectively). A schematic overview of the putative
IAM genomic locus in the four Fusarium species is presented in
Fig. 1.
In addition to these Fusarium species, the putative IAM locus
was only found in the recently released genome sequence of Collet-
otrichum graminicola (http://www.broadinstitute.org/annotation/
genome/colletotrichum_group/MultiHome.html), a close relative
of the IAM-producing species C. gloeosporioides (Robinson et al.,
1998). Using the C. graminicola sequences of the putative IaaM
and IaaH genes, we were able to amplify a homologous genomic lo-
cus containing the two putative IAM genes from C. gloeosporioides,
confirming earlier biochemical data on the presence of the IAM
pathway in this species (Robinson et al., 1998). Similar to the
IAM gene cluster in the four Fusarium species, the putative IAM
genes in the two Colletotrichum species are also organized in a head
51
to head orientation and are separated by a non-coding region of
2969 bp (Fig. 1B).
3.2. IAA production in Fusarium sp.
we measured total indoles and IAA levels in culture filtrates of the
different Fusarium species. Relative levels of indoles were esti-
mated by a colorimetric assay after staining with Salkowski re-
agent. All species produced certain amounts of indoles in media
with tryptophan (Table 1). Similar levels were recorded for all
strains, except for F. proliferatum ET1, which produced significantly
higher levels of indoles compared with all other species. In media
supplemented with tryptophan, the intermediate IAM was de-
tected only in F. proliferatum ET1 culture filtrates (Fig. 2 upper pa-
nel), suggesting that the IAM pathway is highly active in this
species. In media without tryptophan, only trace amounts of IAA
were produced by all species, including F. proliferatum ET1 (data
not shown). When IAM was used as a precursor (Fig. 2 lower pa-
nel), IAA accumulated and IAM was depleted in culture filtrates
from F. proliferatum ET1 and to a lower extent from F. verticillioides,
but not in the filtrates from F. oxysporum and F. fujikuroi. These re-
sults demonstrate the activity of both enzymes of the IAM pathway
(tryptophan monooxygenase and IAM hydrolase) in F. proliferatum
ET1. The accumulation of IAA in IAM-amended F. verticillioides cul-
ture filtrate indicates activity of the IAM hydrolase in this fungus.
IAM could not be detected in F. verticillioides culture filtrates when
tryptophan was added as precursor, suggesting that enzymatic
activity is below detection levels or the enzyme might be non-
functional in this species.
3.3. Enzyme activity assay
In order to verify activity of the IAM-pathway enzymes in F. pro-
liferatum ET1 we conducted an in vitro assay for conversion of tryp-
tophan and IAM by an enzymatic extract obtained from F.
proliferatum mycelium. The fungus was cultured in three different
types of media (CD, EMS and PDB), mycelium was collected and
proteins were extracted under non-denaturing conditions. Protein
extracts were incubated in assay medium with tryptophan or IAM,
and amounts of IAM and IAA were determined after 20 h. IAA was
produced by extracts of mycelia obtained from all types of media.
Much higher amounts were produced with IAM as a precursor
compared with very low levels when tryptophan was used as the
precursor (Fig. 3). Some conversion of IAM to IAA was noticed in
the control (protein-free buffer). A very faint band of IAM was ob-
served after 6 h when tryptophan was used as a precursor (data not
shown), whereas no IAM was visible in the reaction medium after
20 h of incubation with tryptophan (Fig. 3). These results confirm
activity of the IAM pathway in F. proliferatum ET1 and indicate
stronger activity of the IAM hydrolase, the second enzyme which
converts IAM to IAA, than of the first enzyme, tryptophan monoox-
ygenase, which catalyzes conversion of tryptophan to IAM. Thus,
the first step catalyzed by tryptophan monooxygenase might be
the rate limiting step in this pathway of IAA biosynthesis.
3.4. Characterization of knockout and over-expression mutant strains
In order to verify that the identified locus encodes the IAM
pathway genes, we deleted the entire locus (both reading frames
and the intergenic region) in F. proliferatum ET1 by targeted gene
replacement. The wild-type and Diaam/Diaah mutant strains were
cultured in media with tryptophan or IAM, indoles were extracted
from the culture filtrate, and the levels of IAM and IAA were deter-
mined by HPLC analysis. The deletion mutants showed a drastic
reduction in IAA levels in media with tryptophan (Table 2). A very
52 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57

(A)

(B)

Fig. 1. Organization of the IAM genes locus in four Fusarium species and two Colletotrichum species. Sequence of F. verticillioides, F. oxysporum and C. graminicola were
obtained from the BROD database. IAM loci from F. proliferatum and F. fujikuroi were isolated and sequenced during this study. The sequence of C. gloeosporioides IAM locus
was obtained from a draft of genomic sequence (courtesy of Dov Prusky, unpublished data).

Table 1
Total indoles production by Fusarium species.

Species Indoles (OD540, lg/ml)

24 h 32 h 48 h 72 h
Trp +Trp Trp +Trp Trp +Trp Trp +Trp
F. oxysporum 4287 0.09 ± 0.01 4.15 ± 0.31 0.42 ± 0.06 6.30 ± 0.81 1.37 ± 0.39 7.85 ± 1.01 0.46 ± 0.08 10.54 ± 1.32
F. proliferatum ET1 0.36 ± 0.05 8.26 ± 0.27 0.60 ± 0.10 15.83 ± 2.77 1.23 ± 0.15 29.47 ± 2.24 1.05 ± 0.32 46.95 ± 3.6
F. fujikuroi m567 0.23 ± 0.05 2.46 ± 0.11 0.45 ± 0.03 4.70 ± 0.42 1.60 ± 0.30 6.89 ± 0.19 2.06 ± 1.03 16.84 ± 0.92
F. verticillioides 149 0.14 ± 0.08 5.57 ± 0.20 0.47 ± 0.06 8.62 ± 0.96 1.96 ± 0.28 10.72 ± 0.80 1.32 ± 0.15 8.90 ± 0.58
C. gloeosporioides 3.1.3 0.23 ± 0.04 19.98 ± 1.92 0.28 ± 0.06 23.77 ± 1.89 0.41 ± 0.06 30.80 ± 2.81 0.91 ± 0.09 50.37 ± 2.80

Indoles were extracted and stained with Salkowski’s reagent; absorbance was measured at OD540, and indoles amounts (lg/ml) were calculated according to absorbance of
known IAA concentrations. The values are means of three replicates. ± standard deviation.

Fig. 2. Production of IAA from tryptophan and IAM by four Fusarium species. Fungi
were cultured in CD medium with 4 mM tryptophan or 0.5 mM IAM. Culture
filtrates were sampled every 24 h and total indoles were extracted and analyzed by
TLC.
Fig. 3. Enzyme activity assay of extracts from F. proliferatum ET1. The fungus was
cultured in different media, mycelia were harvested after 72 h of cultivation and
proteins were extracted. The protein extracts were incubated for 16 h with either
tryptophan or IAM and then indoles were extracted and analyzed by TLC. CD –
Chapex Dox medium, EMS – Ehmerson’s YPSs medium, PDB – potato dextrose
broth, IAM – assay mix containing IAM without protein extract, Tryp – assay mix
containing tryptophan without protein extract, Std – standard IAA and IAM.
 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
small peak corresponding to the retention time of IAM was re-
corded in culture media of the mutant strain. However, TLC analy-
sis showed that there was no spot of IAM in extracts from these
cultures, unlike the extracts from the wild type strain in which a
clear IAM spot was observed (Fig. S2). Furthermore, when IAM
was added to cultures of the mutant, it was retained in the medium
and was not converted to IAA, in contrast to conversion of IAM to
IAA in the wild type cultures. Similar results were obtained by an
in vitro enzymatic assay (data not shown). These results confirm
that the identified locus encodes the IAM pathway genes in F. pro-
liferatum ET1. The corresponding gene were therefore designated
IAM1 (JN166967) and IAH1 (JN166966), for F. proliferatum IaaM
and IaaH, respectively.
To further confirm the function of the two genes we over-ex-
pressed them in F. fujikuroi (which produces only trace amounts
of IAA and does not produce any IAM). We generated transgenic
strains expressing either the IAH1 gene alone (seven independent
isolates, represented by strain T1), or both the IAH1 and the IAM1
(three independent isolates, represented by strain T2). The trans-
genic strains were cultured in media with tryptophan or IAM, the
relative amounts of indoles were determined by Salkowski reac-
tion and IAM and IAA amounts were determined by TLC and HPLC
analyses. In media with IAM, all the transgenic strains produced in-
creased amounts of indoles compared to the F. fujikuroi wild type
strain (Table 3). In contrast, when tryptophan was used as a pre-
cursor, only the three strains containing both genes (represented
by strain T2), produced elevated levels of indoles. The transfor-
mants containing only the IAH1 gene (represented by strain T1),
produced wild type levels of indoles (Table 3). In accordance with
these results, TLC analyses showed that the T2 strain produced ele-
vated levels of IAA also in media with tryptophan (Fig. S3). Quan-
titative HPLC analysis confirmed that in medium with IAM both
the T1 and T2 transformants produced elevated levels of IAA,
whereas tryptophan was converted to IAA only by strain T2, which
produces increased amounts of both IAM and IAA (Table 2).
3.5. Analysis of IAA gene expression
Expression levels of the IAM and IAH genes were estimated by
semi-quantitative PCR on cDNA samples from the wild type strains
and the F. fujikuroi transgenic strains carrying the IAH1 (T1) or both
IAH1 and IAM1 (T2) over-expression vectors, pOE-IaaH and pOE-
IaaM. As expected, expression levels of the IAM1 gene were highly
elevated in strain T2, whereas expression levels of the IAH1 gene
were elevated in strains T1 and T2 (Fig. 4A). Northern blot analysis
confirmed these results and also indicated that expression levels of
both genes in the four Fusarium species were very low or below
detection levels (Fig. 4B). Hybridization bands corresponding to
the transcripts of both genes were detected in F. proliferatum and
F. verticillioides, although in each species one of the genes was more
Table 2
HPLC analysis of IAA, IAM and TOL in wild type and mutant strains.
Species/strain Tryptophan (4 mM)
lM IAM
F. proliferatum ET1 23.0 ± 1.4
F. verticillioides 149 7.6 ± 0.4
F. oxysporum 4287 15.2 ± 1.5
F. fujikuroi m567 9.0 ± 1.2
F. proliferatum #612 6.5 ± 0.5
F. fujikuroi #T1 9.5 ± 0.9
F. fujikuroi #T2 224.2 ± 8.6
Extracts were separated by HPLC and quantified by comparison of peak area with area of known quantities of IAA, IAM and TOL samples. F. proliferatum #612 is a DiaaM/
DiaaH double mutant, F. fujikuroi T1 is a transgenic strain over-expressing the F. proliferatum ET1 IAH1 gene, F. fujikuroi T2 is a transgenic strain over-expressing both of the F.
proliferatum ET1 IAM1 and IAH1 genes. The values are means of three replicates. ± standard deviation.
53
abundant than the other. Only a weak signal of the IaaH gene was
detected in F. oxysporum, while in F. fujikuroi both transcripts could
not be detected.
The correlation between expression levels of the IAM genes and
IAA production suggested that the low expression levels of the IAM
genes, and particularly of IaaM, might be a limiting factor in IAA
production. Indeed, in media without tryptophan the T2 over-
expression strain produced high levels of indoles (Table 4) includ-
ing significant amounts of IAA (Fig. 5), unlike the wild type strains,
which do not produce IAA in media without tryptophan.
Comparison of the expression levels of both genes in the F. pro-
liferatum ET1 wild-type strain indicated that the IAH1 transcript is
more abundant than that of IAM1, suggesting that the intergenic
promoter region has a stronger transcriptional activity in direction
of IAH1 gene. In order to evaluate activity of the promoter in each
direction, we cloned the complete F. proliferatum ET1 promoter re-
gion in (+) and () orientations between the reading frames of YFP
and dsRED (Fig. S1). Transgenic strains were obtained with each
cassette, fluorescence intensities were recorded in each strain.
The results confirmed a much stronger activity of the promoter
in the minus () orientation (direction of IAH1) (Fig. 6). In order
to calculate the relative activity of the promoter in each direction,
we measured fluorescence intensity in the red and yellow channels
of fluorescence microscope in 50 spores from each strain, and cal-
culated the relative activity of the promoter in each direction. A set
of equations was developed in order to normalize the protein spe-
cific activity (YFP versus DsRED) and the impact of the different ge-
netic background (strains #1 and #2), as described in Material and
Methods. According to these calculations, the promoter activity in
direction of the IAH1 gene () is about three times stronger than in
direction of the IAM1 gene (+).
These results show that IAA production is determined by tran-
scription levels of both genes, and that the flow of the internal
tryptophan pool can be directed to the IAM pathway by up-regula-
tion of the IAM1 gene.
3.6. Sequence analysis of IAH and IAM genes from different Fusarium
species
From the four different Fusarium species with identified IAM
genes, only F. proliferatum ET1 expresses both genes in detectable
amounts and produces significant levels of IAA. Sequence compar-
ison of the genomic loci containing the IAM genes in the four Fusar-
ium species revealed that the region in F. oxysporum as presented in
the genome database at BROD is about 1 kb shorter compared to
the other species (Fig. 7). We sequenced the entire IAM locus in
F. oxysporum and found that the IAM1 gene (FOXG_02584) is
wrongly annotated in the published genome database: the gene
actually contains the 50 part, however, the start codon is missing.
In addition, the sequence contains several mutations that result
IAM (0.5 mM)
lM TOL lM IAA lM TOL lM IAA
5.6 ± 0.3 78.7 ± 4.5 4.1 ± 0.2 105.1 ± 3.4
6.2 ± 0.5 10.2 ± 0.7 4.2 ± 0.3 76.3 ± 4.7
9.9 ± 1.2 15.7 ± 0.9 6.1 ± 0.2 18.9 ± 2.0
3.7 ± 0.2 9.3 ± 0.9 3.4 ± 0.2 14.1 ± 0.8
6.0 ± 0.4 12.3 ± 1.3 2.7 ± 0.5 11.8 ± 0.5
5.4 ± 0.4 10.4 ± 0.7 6.0 ± 0.3 150.6 ± 11.7
24.4 ± 1.7 1459.4 ± 128.3 5.6 ± 0.2 225.4 ± 7.5
54 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57

Table 3
Total indoles production by F. fujikuroi m567 transgenic strains over-expressing F. proliferatum ET1 IAM biosynthesis genes.

Species Indoles (lg/ml)

24 h 48 h 72 h
+Trp +IAM +Trp +IAM +Trp +IAM
F. proliferatum ET1 35.10 ± 2.15 44.46 ± 4.10 47.70 ± 2.82 63.84 ± 3.86 53.81 ± 4.10 58.97 ± 4.19
F. fujikuroi m567 4.79 ± 0.37 17.14 ± 0.93 5.15 ± 0.53 26.99 ± 2.13 4.56 ± 0.33 14.59 ± 0.09
F. fujikuroi #T1 4.88 ± 0.19 47.20 ± 2.30 5.47 ± 0.55 58.09 ± 4.27 5.02 ± 0.27 64.75 ± 3.27
F. fujikuroi #T2 65.98 ± 3.80 56.91 ± 4.72 118.01 ± 7.80 99.13 ± 2.86 154.86 ± 8.76 129.82 ± 6.77

Fungi were incubated in media with either 4 mM tryptophan (+Trp) or 0.5 mM IAM (+IAM), indoles were extracted, stained with Salkowski reagent, absorbance was
measured at OD540 and amount of indoles (lg/ml) was calculated by comparing with absorbance of known IAA concentrations. F. fujikuroi T1 and T2 respectively, represent
two types of transgenic strains; one that produces large amounts of indoles in media with IAM (represented by strain T1) and one that produces large amounts of indoles in
media with either IAM or Tryp (represented by strain T2). The values are means of three replicates. ± standard deviation.

Fig. 5. Production of IAA without external supply of tryptophan. Fungi were

cultivated in CD medium without tryptophan and indoles were extracted from the
culture filtrates at 24 and 48 h. F.v – F. verticillioides 149, F.p – F. proliferatum ET1,
F.o – F. oxysporum 4287, Ff – F. fujikuroi m567, T1 – F. fujikuroi m567 transgenic
strain expressing the IAH1 gene, T2 – F. fujikuroi m567 transgenic strain expressing
the F. proliferatum IAM1 and IAH1 genes.

affect the promoter activity. Collectively, these results suggest that

the F. oxysporum and F. fujikuroi IAM pathways are non-functional
Fig. 4. Gene expression in wild type and transgenic strains. (A) qRT-PCR. Fungi
due to a series of defects in the genes as well as in the regulatory
were cultured in CD + 4 mM tryptophan, mycelia were harvested after 48 h, RNA region. In F. verticillioides, two amino acids insertions in the IAM1
was extracted and RT-PCR was performed. Samples were analyzed after 35 cycles. ORF might have a negative effect on the IAM activity, since no
Arrows indicate location of faint bands. M – IaaM, H – IaaH. (B) Northern blot. RNA IAM can be detected following feeding of tryptophan, and signifi-
samples were separated on a gel, blotted and probed with 32P labeled DNA
cant IAA production is obtained only after IAM feeding.
fragments of IaaH, IaaM and ribosomal RNA. F.v – F. verticillioides 149, F.p – F.
proliferatum ET1, F.o – F. oxysporum 4287, Ff – F. fujikuroi m567, T1 – F. fujikuroi
m567 transgenic strain expressing the IAH1 gene, T2 – F. fujikuroi m567 transgenic
strain expressing the F. proliferatum IAM1 and IAH1 genes. 3.7. Phylogeny of the fungal IAM genes

The two fungal IAM genes are situated next to each other, sim-
ilar to the bacterial IAM genes. In addition, in all the fungi studied
so far the genes are organized in a head to head orientation (Fig. 1).
Table 4
Indoles production by transgenic strain in media without external tryptophan.
In order to determine the distribution of the IAM genes across the
fungal kingdom, we performed a megablast search of the available
Species/strain Indoles (lg/ml)
fungal genomes with the predicted F. proliferatum IAM1 and IAH1
24 h 48 h 72 h 96 h proteins. Homologs of both genes were identified in several species
F. proliferatum ET1 0.12 ± 0.01 0.55 ± 0.03 0.73 ± 0.02 1.05 ± 0.07 other than Fusarium and C. graminicola. However, in these species
F. fujikuroi m567 0.00 0.00 0.20 ± 0.05 0.42 ± 0.05 the IAM gene was not associated with an IAH gene in the same ge-
F. fujikuroi #T1 0.00 0.41 ± 0.03 0.64 ± 0.02 0.70 ± 0.04 netic locus (Fig. 8).
F. fujikuroi #T2 1.20 ± 0.08 3.56 ± 0.07 5.11 ± 0.06 7.66 ± 0.23
The IAM hydrolase encoded by IAH belongs to a large family of
F. fujikuroi T1 and T2 are transgenic strains expressing either the F. proliferatum ET1 amidases that are highly abundant in fungi. In contrast to trypto-
IAH1 (strain T1) and IAH1 and IAM1 (strain T2). The values are means of three phan monooxygenase, which is distinct from other monooxygena-
replicates. ± standard deviation.
ses, IAH is highly homologous to several IAA-unrelated amidases.
Nevertheless, IAH forms a clade that is distinguished from other
amidase-encoding genes, and it is present in those species in which
in frame shifts and formation of early stop codons. These signifi- both the IAM and IAH genes were discovered (next to each other in
cant sequence aberrations and mutations explain the lack of tryp- a head to head orientation), as well as in several other species, in
tophan monooxygenase activity in F. oxysporum. which the IAM gene is missing (Fig. 9). Interestingly, one class of
In F. fujikuroi the 50 end of the IAM gene contains a deletion of fungal amidaseswith domain pfam0142, which are present only
40 bp, which results in a frame shift. Furthermore, although overall in fungi of the genus Claviceptacea, groups together with bacterial
homology between the F. fujikuroi and F. proliferatum intergenic re- IaaH in the same clade, hinting that the origin of IaaH in this genus
gion is 97%, the F. fujikuroi sequence in this region contains a dele- (eg., Claviceps purpurea and Epichloe festuca) was by a horizontal
tion, an insertion, and a few nucleotide exchanges that possibly gene transfer from Agrobacterium of a similar bacterium.
 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57 55

Fig. 6. Analysis of the IAM1/IAH1 dual promoter. F. proliferatum ET1 was transformed with two plasmids each containing the IAM1/IAH1 dual promoter between the YFP and
DsRed genes, in two orientations (as shown on the left, also see Fig. S1). The arrow points to the direction of the IAH1 gene in the native locus (see Fig. 1A). Pictures were taken
using same exposure time in both filters, so intensity is comparable. From left to right: YFP filter (a and e), Rhodamine filter (b and f), DIC (c and g), merged images of red,
yellow and DIC channels (d and h). Bars = 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Comparison of IaaH/IaaM regions in the four Fusarium species. MUSCLE alignments of the IaaH/IaaM protein sequences and the bidirectional promoter between IaaH
and IaaM in F. proliferatum, F. fujikuroi, F. oxysporum and F. verticillioides are schematically shown. Boxes represent protein sequences, lines nucleic acid sequences.
Homologous regions are indicated by different gray colors. (M) – methionine, () – stop codon, (4) – location of deletion (+) – insertion, (fr) – frame shift.

Fig. 8. Phylogeny of tryptophan monooxygenase in fungi and bacteria. Sequence alignment was done by MUSCLE, curation with the built-in curer, phylogeny with PhyML and
tree rendering with TreeDyn (bootstrap = 500). See Table S2 for accession numbers of the sequences and for species names. Branch support values are shown.
56 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
4. Discussion
While many fungi have the capacity to produce IAA in axenic
cultures, synthesis of IAA via the bacterial IAM pathway has been
previously reported only in Colletotrichum sp. (Robinson et al.,
1998). However, the Colletotrichum genes could not have been
identified until now due to the lack of genomic sequence in this
fungus. Here we showed for the first time that the IAM pathway
genes are present in a number of Fusarium species. The two genes
were also found in the recently published C. graminicola genome as
well as in C. gloeosporioides, confirming the previous biochemical
data (Robinson et al., 1998).
Among the different Fusaria that we have examined, F. grami-
nearum and Fusarium solani, both not belonging to the G. fujikuroi
species complex (GFC), did not contain the IAM genes. Interest-
ingly, the pathways for two other phytohormones, gibberellins
and cytokinines, which are widespread in Fusarium species of the
GFC are also missing in F. graminearum and F. solani. While the
functional significance of this finding is yet unclear, it hints that
the IAM genes found in species belonging to the GFC might have
originated in the genus Fusarium by a gene transfer, possibly from
bacteria. More work is needed to determine whether indeed this is
the case, but if a horizontal gene transfer had taken place, it would
have been after the divergence of GFC species complex from other
Fusaria, e.g., F. graminearum and F. solani.
Of the four Fusarium species in which the IAM genes were
found, only F. proliferatum had a functional pathway, while F. ver-
ticillioides showed activity of the IAM hydrolase. In F. fujikuroi
and F. oxysporum the pathway is inactive due to mutations in the
IaaM genes and possibly also due to very weak or lack of gene
expression. Transcription analyses revealed significant differences
in expression of the two genes. In F. proliferatum expression of
the IAH1 gene was much stronger, whereas in F. verticillioides
expression of the IAM1 gene was higher. Thus, the intergenic re-
gion between the two genes has an asymmetric promoter activity.
Quantitative analysis using YFP- and DsRed promoter fusions indi-
cated that in F. proliferatum transcription is about 3-fold stronger in
Fig. 9. Phylogeny of IAM hydrolase and related amydases in fungi and bacteria. Support values are presented next to each branch point. The alignment was done by MUSCLE,
curation with the built-in curer, phylogeny with PhyML and tree rendering with TreeDyn (bootstrap = 500). See Table S2 for accession numbers of the sequences.
direction of IAH1. Nevertheless, the normal transcript levels of
IAM1, although relatively low, are sufficient to support IAA produc-
tion, as shown by accumulation of both IAM and IAA in F. prolifer-
atum ET1 cultures. Further analyses will be necessary to define the
regulatory elements in the promoter region responsible for expres-
sion of both genes.
Normally, external addition of tryptophan is necessary for IAA
synthesis in axenic fungal cultures, suggesting that there is a cor-
relation between expression levels of the first enzyme in the path-
way and the amount of IAA produced. To address this question we
generated F. fujikuroi transgenic strains that over-expressed the F.
proliferatum IAM genes and studied IAA production in media with
and without tryptophan. In media with tryptophan, the transgenic
strains expressing both F. proliferatum genes accumulated about 10
and 20-fold more IAM and IAA, respectively (Table 3). In media
with IAM they accumulated only two fold more IAA, similar to pro-
duction levels by a strain expressing only the IAH1. Thus, the lim-
iting factor in IAA production is transcription level of the IaaM gene
and subsequently activity of the tryptophan monooxygenase en-
zyme. Furthermore, the transgenic strains also produce significant
amounts of IAA even in media without tryptophan. This is in con-
trast to numerous studies, all of which reported that external tryp-
tophan was necessary for IAA production in fungal axenic cultures
(reviewed by Tudzynski and Sharon, 2001). Our results suggest
that tryptophan flux into IAA biosynthesis is determined by
expression level of the IaaM gene, and accordingly, by activity level
of tryptophan monooxygenase. It is possible that in fungi that do
not have the IAM pathway genes, the flux of tryptophan to the
alternative pathways might be regulated in a similar way (see
graphical abstract for the different pathways).
The physiological role of IAA production in fungi is unclear. Un-
der laboratory conditions, the F. proliferatum mutant strains in
which the IAM genes were deleted had no detectable phenotype
(data not shown). Similar results were reported for deletion of
the gibberellin biosynthesis genes in F. fujikuroi (Tudzynski,
2005). However, we could not test the effect of gene deletion on
plant colonization by F. proliferatum since the ET1 was originally
 E. Tsavkelova et al. / Fungal Genetics and Biology 49 (2012) 48–57
isolated from orchid, where it is assumed to exist as an endophyte
(Tsavkelova et al., 2003, 2008). Interestingly, this strain was also
shown to produce gibberellins, in contrast to maize pathogenic
F. proliferatum strains, which normally do not produce this class of
phytohormones (Malonek et al., 2005a, 2005b, 2005c; Tsavkelova
et al., 2008). These results indicate a possible connection between
the endophytic lifestyle and production of phytohormones. The
only evidence for in planta production of IAA known so far is in
C. gloeosporioides (Maor et al., 2004), which has an active IAM path-
way and in which we have now identified the IAM genes. This fun-
gus requires tryptophan for the production of IAA in axenic culture
however, it also produces significant levels of IAM in infected
plants. Thus, production of IAA (and possibly of other plant hor-
mones) might be important at certain stages of host colonization
in some species but not in others, depending on host plant, the
infection strategy, and pathogenic versus endophytic lifestyle of
the fungus.
Acknowledgments
We thank Dov Prusky for kindly providing us the sequence of
the C. gloeosporioides IAM gene cluster, Sabine Huber and Katja
Schäfer for technical assistance, Tal Pupko for assistance in calcula-
tion of relative activities of the IAM promoter, and Antonio Di Pie-
tro for providing the F. oxysporum strain.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fgb.2011.10.005.
References
Barhoom, S., Sharon, A., 2004. CAMP regulation of pathogenic and saprophytic
fungal spore germination. Fungal Genet. Biol. 41, 317–326.
Church, G.M., Gilbert, W., 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81,
1991–1995.
Costacurta, A., Vanderleyden, J., 1995. Synthesis of phytohormones by plant
associated bacteria. Crit. Rev. Microbiol. 21, 1–18.
Di Pietro, A., Roncero, M.I.G., 1998. Cloning, expression, and role in pathogenicity of
pg1 encoding the major extracellular endopolygalacturonase of the vascular
wilt pathogen Fusarium oxysporum f. sp. lycopersici. Mol. Plant–Microbe Interact.
11, 91–98.
Ehmann, A., 1977. The van urk-Salkowski reagent – a sensitive and specific
chromogenic reagent for silica gel thin-layer chromatographic detection and
identification of indole derivatives. J. Chromatogr. 132, 267–276.
Glickman, E., Dessaux, Y., 1995. A critical examination of the specificity of the
Salkowski reagent for indolic compounds produced by phytopathogenic
bacteria. Appl. Environ. Microbiol. 61, 793–796.
Gordon, S.A., Weber, R.P., 1951. Colorimetric estimation of indole acetic acid. Plant
Physiol. 26, 192–195.
Hoff, B., Kück, U., 2005. Use of bimolecular fluorescence complementation to
demonstrate transcription factor interaction in nuclei of living cells from the
filamentous fungus Acremonium chrysogenum. Curr. Genet. 47, 132–138.
Leslie, J.F., Summerell, B.A., 2006. The Fusarium Laboratory Manual. Wiley-
Blackwell.
57
Malonek, S., Rojas, M.C., Hedden, P., Gaskin, P., Tudzynski, B., 2004. The NADPH:
cytochrome P450 reductase gene from Gibberella fujikuroi is essential for
gibberellin biosynthesis. J. Biol. Chem. 279, 25075–25084.
Malonek, S., Bömke, C., Bornberg-Bauer, E., Rojas, M.C., Hedden, P., Hopkins, P.,
Tudzynski, B., 2005a. Distribution of gibberellin biosynthetic genes and
gibberellin production in the Gibberella fujikuroi species complex.
Phytochemistry 66, 1296–1311.
Malonek, S., Rojas, M.C., Hedden, P., Gaskin, P., Hopkins, P., Tudzynski, B., 2005b.
Functional characterization of two cytochrome P450 monooxygenase genes,
P450-1 and P450-4, of the gibberellic acid gene cluster in Fusarium proliferatum
(Gibberella fujikuroi MP-D). Appl. Environ. Microbiol. 71, 1462–1472.
Malonek, S., Rojas, M.C., Hedden, P., Hopkins, P., Tudzynski, B., 2005c. Restoration of
gibberellin production in Fusarium proliferatum by functional complementation
of enzymatic blocks. Appl. Environ. Microbiol. 71, 6014–6025.
Mano, Y., Nemoto, K., Suzuki, M., Seki, H., Fujii, I., Muranaka, T., 2010. The AMI1 gene
family: indole-3-acetamide hydrolase functions in auxin biosynthesis in plants.
J. Exp. Bot. 61, 25–32.
Maor, R., Haskin, S., Kedmi-Levi, H., Sharon, A., 2004. Biosynthesis, regulation and in
planta auxin production by Colletotrichum gloeosporioides f. sp. aeschynomene.
Appl. Environ. Microbiol. 69, 1695–1701.
Normanly, J., 2010. Approaching cellular and molecular resolution of auxin
biosynthesis and metabolism. Cold Spring Harb. Perspect. Biol. 2, a001594.
Oren, L., Ezrati, S., Liberman, Z., Cohen, D., Sharon, A., 2003. Early events in the
Fusarium verticillioides–maize interaction characterized by using a green
fluorescent protein-expressing transgenic isolate. Appl. Environ. Microbiol. 69,
1695–1701.
Patten, C., Glick, B., 1996. Bacterial biosynthesis of indole-3-acetic acid. Can. J.
Microbiol. 42, 207–220.
Reineke, G., Heinze, B., Schirawski, J., Büttner, H., Kahmann, R., Basse, C., 2008.
Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and
its relevance for increased IAA levels in infected tissue and host tumor
formation. Mol. Plant Pathol. 9, 339–355.
Robinson, M., Riov, J., Sharon, A., 1998. Indole-3-acetic acid biosynthesis in
Colletotrichum gloeosporioides f. sp. aeschynomene. Appl. Environ. Microbiol.
64, 5030–5032.
Spaepen, S., Vanderleyden, J., Remans, R., 2007. Indole-3-acetic acid in microbial
and microorganism–plant signaling. Microbiol. Rev. 31, 425–448.
Teichert, S., Schonig, B., Richter, S., Tudzynski, B., 2004. Deletion of the Gibberella
fujikuroi glutamine synthetase gene has significant impact on transcriptional
control of primary and secondary metabolism. Mol. Microbiol. 53, 1661–1675.
Tsavkelova, E.A., Alexandrova, A.V., Cherdyntseva, T.A., Kolomeitseva, G.L., Netrusov,
A.I., 2003. Fungi associated with orchid roots in greenhouse conditions. Mycol.
Phytopathol. (Russ.) 37, 57–63.
Tsavkelova, E.A., Klimova, Y.S., Cherdyntseva, T.A., Netrusov, A.I., 2006. Microbial
producers of plant growth stimulators and their practical use: a review. Appl.
Biochem. Microbiol. 42, 133–143.
Tsavkelova, E.A., Bömke, C., Netrusov, A.I., Weiner, J., Tudzynski, B., 2008. Production
of gibberellic acids by an orchid-associated Fusarium proliferatum strain. Fungal
Genet. Biol. 45, 1393–1403.
Tudzynski, B., Sharon, A., 2001. Biosynthesis, biological role and application of
fungal phytohormones. In: Osiewacz, H.D. (Ed.), The Mycota, Industrial
Applications, vol. 10. Springer-Verlag, Berlin, pp. 183–211.
Tudzynski, B., Mende, K., Weltring, K.M., Kinghorn, J.R., Unkles, S.E., 1996. The
Gibberella fujikuroi niaD gene encoding nitrate reductase: isolation, sequence,
homologous transformation and electrophoretic karyotype location.
Microbiology 3, 533–539.
Tudzynski, B., 2005. Gibberellin biosynthesis in fungi: genes, enzymes, evolution,
and impact on biotechnology. Appl. Microbiol. Biotechnol. 66, 597–611.
Xu, J.-R., Peng, Y.L., Dickman, M.B., Sharon, A., 2006. The dawn of fungal pathogen
genomics. Annu. Rev. Phytopathol. 44, 337–366.
Zhao, Y., Hull, A.K., Gupta, N.R., Goss, K.A., Alonso, J., Ecker, J.R., Normanly, J., Chory,
J., Celenza, J.L., 2002. Trp-dependent auxin biosynthesis in Arabidopsis:
involvement of cytochrome P450s CYP79B2 and CYP79B3. Genet. Dev. 16,
3100–3112.