Microbiology (2011), 157, 2658–2669
DOI 10.1099/mic.0.052506-0
HXK1 regulates carbon catabolism, sporulation,
fumonisin B1 production and pathogenesis in
Fusarium verticillioides
Hun Kim,1,2 Jonathon E. Smith,1 John B. Ridenour,1 Charles P. Woloshuk2
and Burton H. Bluhm1
Correspondence
1
Burton H. Bluhm
2
[email protected]
Received 21 February 2011
Revised 9 June 2011
Accepted 27 June 2011
Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA
In Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are
regulated in response to the types and amounts of carbohydrates present in maize kernels. In this
study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth,
development and pathogenesis. A deletion mutant (Dhxk1) of HXK1 was not able to grow when
supplied with fructose as the sole carbon source, and growth was impaired when glucose,
sucrose or maltotriose was provided. Additionally, the Dhxk1 mutant produced unusual swollen
hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the
Dhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On
maize kernels, the Dhxk1 mutant was substantially less virulent than the wild-type, but virulence on
maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific
differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on
fungal metabolites produced during colonization of maize kernels; the Dhxk1 mutant produced
approximately 50 % less trehalose and 80 % less fumonisin B1 (FB1) than the wild-type. The
reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic
stress in the Dhxk1 mutant. In summary, this study links early events in carbohydrate sensing and
glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new
foothold from which the genetic regulatory networks that underlie pathogenesis and
mycotoxigenesis can be unravelled and defined.
INTRODUCTION
Fusarium verticillioides (Sacc.) Nirenberg (teleomorph
Gibberella moniliformis Wineland) is a ubiquitous pathogen of maize and is endemic virtually everywhere maize is
grown in the world (Nelson et al., 1993). F. verticillioides is
remarkably versatile, capable of infecting seedlings, roots,
stalks and ears of maize plants. Although yield losses can be
considerable due to poor stand establishment (seedling
infections) and lodging before harvest (stalk infections),
kernel infections present a unique set of problems for
agricultural production due to the accumulation of
fumonisin mycotoxins in colonized kernels. At least 28
fumonisin analogues have been identified, the B series of
which is most commonly associated with F. verticillioides
(Rheeder et al., 2002); of these, fumonisin B1 (FB1) is
predominant during kernel infections and is also the most
toxic to humans and livestock (Desai et al., 2002).
Abbreviation: FB1, fumonisin B1.
Three supplementary figures and two supplementary tables are available
with the online version of this paper.
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Ingestion of FB1 leads to the obstruction of sphingolipid
biosynthesis by inhibiting ceramide synthase (sphinganine
N-acyltransferase) (Desai et al., 2002), and results in
leukoencephalomalacia in horses and oesophageal cancer
and birth defects in humans (Hendricks et al., 1999).
The biochemical and genetic basis of fumonisin biosynthesis
is increasingly well understood. Fumonisins are derived
from polyketides, the backbone chains of which are
elongated through incorporation of acetyl-CoA or malonyl-CoA via Claisen condensations, followed by a series of
condensations, reductions and esterifications that result in
the formation of structural analogues within a series (Gerber
et al., 2009). In F. verticillioides, fumonisin biosynthetic
(FUM) genes are physically clustered within the genome and
have been studied extensively. Currently, 17 FUM genes are
known to constitute the fumonisin biosynthetic gene cluster
(Brown et al., 2007; Proctor et al., 2003): genes encoding
proteins similar to a polyketide synthase (FUM1), cytochrome
P450 monooxygenases (FUM6, FUM12, FUM15), dehydrogenases (FUM7, FUM13), an aminotransferase (FUM8), a
dioxygenase (FUM9), fatty acyl-coenzyme A synthetases
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Role of HXK1 in pathogenesis of F. verticillioides
(FUM10, FUM16), a tricarboxylate transporter (FUM11), a
peptide synthetase (FUM14), longevity assurance factors
(FUM17, FUM18), an ABC transporter (FUM19), a putative
pathway-specific transcription factor (FUM21), and a gene
of unknown function (FUM20). In addition, a small number
of regulatory genes have been identified in F. verticillioides
that affect FB1 biosynthesis, including FCC1 (a C-type
cyclin), PAC1 (a pH-responsive transcription factor gene),
ZFR1 (a zinc finger transcription factor gene) and AREA (a
nitrogen metabolism regulatory gene) (Flaherty et al., 2003;
Flaherty & Woloshuk, 2004; Kim & Woloshuk, 2008; Shim
& Woloshuk, 2001). However, genes that specifically
regulate the induction of FB1 biosynthesis in response to
the plant environment have not been described.
Important environmental cues have been identified that
modulate the genetic regulatory network underlying
fumonisin biosynthesis. For example, the sources of carbon
available to F. verticillioides play a critical role in the
induction of fumonisin biosynthesis. The availability of
amylopectin, the major constituent of starch in maize
kernels, correlates strongly with high levels of FB1
biosynthesis during kernel colonization. FB1 biosynthesis
increases dramatically with kernel maturity, peaking when
starch accumulates in endosperm tissues during kernel
development (Bluhm & Woloshuk, 2005; Warfield &
Gilchrist, 1999), and is substantially higher in the starchrich endosperm of mature kernels than the lipid- and
protein-rich germ tissues (Shim et al., 2003). Additionally,
FB1 biosynthesis is repressed by nitrogen sources such as
ammonium and glutamine (Kim & Woloshuk, 2008; Shim
& Woloshuk, 1999), and high pH (Flaherty et al., 2003).
The ability of diverse environmental cues to influence
fumonisin biosynthesis hints at the existence of a complex
genetic regulatory network in F. verticillioides.
In F. verticillioides, the signal transduction networks that
regulate FB1 biosynthesis in response to carbohydrate
availability have not been elucidated at the molecular level.
Of critical importance is to understand how F. verticillioides perceives and utilizes starch and other carbohydrates
during kernel colonization. Comparative analyses of gene
expression in F. verticillioides during growth on germ
versus endosperm tissues have led to the identification of
FST1, which is predicted to encode a member of the major
facilitator superfamily (MFS), homologous to sugar
transporters in other filamentous fungi (Bluhm et al.,
2008). Expression of FST1 is induced on endosperm tissue
and regulated by ZFR1, a gene encoding a zinc finger
transcription factor that is required for FB1 biosynthesis
(Bluhm et al., 2008). Disruption of FST1 causes a severe
reduction in FB1 biosynthesis and virulence. Intriguingly,
the protein encoded by FST1 may not function as a
transporter, but rather as a carbohydrate sensor, analogous
to RCO3 in Neurospora crassa (Kim & Woloshuk, 2011).
However, additional mechanisms of carbohydrate detection are likely to exist in filamentous fungi. In higher
eukaryotes, hexokinases are multifunctional enzymes, with
roles in catabolism (e.g. required for the first step of
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glycolysis) and signalling (e.g. glucose sensors) (Harrington
& Bush, 2003; Santangelo, 2006). Although hexokinases
have been implicated in sugar sensing/signalling in plants
and animals (Claeyssen & Rivoal, 2007; Wilson, 2003; Xiao
et al., 2000), the extent to which hexokinases function as
sugar sensors is unknown among filamentous fungi, and
has not been investigated in F. verticillioides.
The purpose of this study was to investigate the potential
function of hexokinase genes in F. verticillioides during
pathogenesis. We focused on HXK1, a gene predicted to
encode a putative hexokinase, whose expression was
reduced significantly in a Dzfr1 strain (Bluhm et al.,
2008). Here, a HXK1 deletion mutant (Dhxk1) was
characterized, and defects were observed in growth,
development, pathogenesis and secondary metabolism,
including FB1 biosynthesis. Several phenotypes were most
evident when fructose was supplied as the sole carbon
source, possibly indicating that HXK1 encodes a fructokinase. Taken together, the results support the hypothesis
that hexokinases such as the HXK1 product serve
important roles in the transduction of external environmental cues that regulate pathogenesis and mycotoxigenesis in F. verticillioides.
METHODS
Fungal strains and media. F. verticillioides strain 7600 (Fungal
Genetics Stock Center) was the wild-type in this study, and Dhxk1
(HXK1 deletion strain) and Dhxk1-comp (strain Dhxk1 complemented with the wild-type copy of HXK1) were created as described
below. All strains were stored at 280 uC in 25 % (v/v) glycerol.
Conidia were obtained from cultures grown on potato dextrose agar
medium (PDA; BD) by rinsing plates with sterile water, and conidia
were quantified with a haemocytometer prior to inoculation. To
extract fungal genomic DNA, strains were grown in yeast extract
peptone (YEP) medium (5 g yeast extract l21, 10 g peptone l21)
containing 20 g glucose l21. To quantify sugar uptake, cultures were
resuspended into defined liquid (DL) medium (per litre: 3 g KH2PO4,
0.3 g MgSO4, 1 g BSA, 5 g NaCl, 20 g of either glucose or fructose)
from YEP medium.
Nucleic acid isolation and analysis. Bacterial plasmids were
isolated with a Qiagen Miniprep DNA purification system. PCR
primers were obtained from Integrated DNA Technologies. Isolation
of protoplasts from F. verticillioides and transformation were
performed as described previously (Shim & Woloshuk, 2001).
Standard procedures were followed for isolation of fungal genomic
DNA and Southern blotting (Kim & Woloshuk, 2008).
Phylogenetic analysis. A dataset of amino acid sequences encoding
putative proteins was assembled from the genome of F. verticillioides
(Broad Institute) by BLASTP (Altschul et al., 1990). Sequences were
initially aligned with CLUSTAL_X (Larkin et al., 2007). Subsequently,
ambiguously aligned regions were removed manually. The amino acid
dataset was analysed by distance and neighbour-joining methods with
a Dayhoff PAM matrix in PHYLIP v3.68 (http://evolution.genetics.
washington.edu/phylip.html). Default settings were used and internal
branch support was evaluated based on 100 bootstrap replicates.
Deletion and complementation of HXK1. To delete HXK1, a DNA
fragment consisting of a selective resistance gene cassette (HPH,
hygromycin B phosphotransferase) fused between the 59 and 39
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H. Kim and others
flanking sequences of HXK1 was generated by double-joint PCR
(Yu et al., 2004). To this end, DNA fragments corresponding to 59
(1.4 kb) and 39 (1.4 kb) regions flanking the ORF of HXK1 were
amplified from genomic DNA with the primer pairs HXK1-1
(59-CCATTTGGCGCGTCAGCAGA-39)/HXK1-2 (59-TTTGTGCTCACCGCCTGGACGATTTGGCGGGGTAGTGT-39) and HXK1-3
(59-GAATAGATGCCGACCGGGAATCTCCCTCACCAGCCTTTTC-39)
/HXK1-4 (59-TCATCATCGAACCCCAGACTTGTA-39), respectively. The HPH cassette was amplified from pCB1003 (Fungal
Genetics Stock Center) with primers HYG5 (59-GTCCAGGCGGTGAGCACAAA-39) and HYG3 (59-TTCCCGGTCGGCATCTATTC-39). Sequences complementary to primers HYG5 and HYG3
were incorporated into the 59 ends of primers HXK1-2 and HXK1-3
(underlined above) to facilitate fusion of the three PCR products.
For double-joint PCR, the 59 flanking region of HXK1, the HPH
cassette and the 39 flanking region of HXK1 were fused as described
by Yu et al. (2004). Finally, the fusion product was amplified with
nested primers HXK1-NF (59-ACCTACAGGACAAGCACCCCACAT-39) and HXK1-NR (59-TCCTCCGATTGCCCAGACGA-39)
to create a 3.6 kb product containing the HPH cassette fused to the
HXK1 flanking regions. The resulting PCR product was concentrated
and transformed into the wild-type strain of F. verticillioides as
described by Kim & Woloshuk (2008). The deletion strains
were confirmed by Southern analysis by probing for either the
absence or the presence of the HXK1 ORF. The probe used for
Southern analysis was amplified with the primer pair HXK1-NSF
(59-TCCCCGAACTGCCTACGATGTGAC-39) and HXK1-NSR (59GCGGCGCCGACACCACT-39). To complement the Dhxk1 mutant,
a PCR product containing the wild-type copy of HXK1 was
amplified with primers HXK1-NF and HXK1-NR from strain
7600, and co-transformed into the Dhxk1 mutant with plasmid
pKS-GEN, which confers resistance to geneticin (Flaherty et al.,
2003). The resulting transformants were screened by PCR with
primers HXK1-NSF and HXK1-NSR for the deletion region, and the
growth of complemented strains was confirmed on a defined
medium containing fructose as the sole carbon source.
Quantification of FB1 and ergosterol during kernel colonization.
Maize kernels (inbred B73) were inoculated as described by Kim &
Woloshuk (2008), and FB1 and ergosterol were quantified after 7 days
growth, as described by Bluhm & Woloshuk (2005). Briefly, FB1 was
extracted from ground maize kernels (500 mg) with 2 ml acetonitrile/
water (1 : 1, v/v). The extracts were passed through equilibrated C-18
solid phase extraction columns (Agilent Technologies), and FB1 was
eluted with 2 ml acetonitrile/water (7 : 3, v/v). Analysis was performed
on an HPLC (Shimadzu Scientific Instruments) equipped with an
analytical C-18 column (15064.6 mm, Alltech) and a fluorescence
detector (excitation 335 nm/emission 440 nm). FB1 was quantified by
comparison of peak areas with those of an FB1 standard (SigmaAldrich). To quantify ergosterol, ground kernels (~500 mg) were
extracted in 2 ml chloroform/methanol (2 : 1, v/v). Ergosterol was
analysed with an HPLC system with a 4.6 U ODS column
(25064.6 mm, Alltech) and a UV detector (Shimadzu) set to measure
absorbance at 282 nm. Quantities were determined by comparing peak
areas of samples to those of a standard curve generated from HPLCgrade ergosterol (Sigma-Aldrich).
Pathogenicity assay. To assess infection and colonization of maize
kernels, seeds of maize inbred B73 were sown and self-pollinated in
greenhouse beds, and ears were removed at the dough stage of kernel
development (growth stage R4, approximately 28 days after
pollination; Ritchie et al., 1993). All procedures including inoculation
and incubation were performed as described by Kim & Woloshuk
(2011). To evaluate infection and colonization of maize stalks, maize
hybrid 33D37 (Pioneer) was grown in the greenhouse and inoculated
when tassels emerged (growth stage VT, approximately 8 weeks after
germination; Ritchie et al., 1993). Fifty microlitres of conidia
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(105 ml21) were injected into internodes of maize stalks with a
syringe attached to a needle. Ear and stalk rot assays were repeated at
least twice, with at least three replications per strain in each
independent experiment.
Quantification of sugar uptake. Total sugar amounts were
measured with a modified anthrone method (Laurentin & Edwards,
2003). Briefly, fungal strains were grown in YEP medium for 3 days,
and mycelia were harvested by centrifugation. Mycelia (0.22 g) from
each strain were then transferred to DL-glucose or DL-fructose
medium, with at least three replications per strain. From each
culture, 50 ml medium was collected at 0, 24, 48, 72 and 96 h, and
diluted 200-fold with water for quantification of sugar. Samples
(300 ml) were mixed with 600 ml anthrone reagent [2 mg anthrone
(ml sulfuric acid)21] in test tubes. After boiling for 5 min, A620 was
measured with a spectrophotometer (Beckman Instruments). A
standard curve was prepared with either glucose or fructose
(Sigma-Aldrich).
Metabolic fingerprinting of colonized kernels. Kernels of inbred
B73 were selected arbitrarily for metabolic fingerprinting at 7 days
after inoculation with the wild-type, Dhxk1 and Dhxk1-comp strains.
Metabolic fingerprinting was performed essentially as described by
Smith & Bluhm (2011), with minor modifications. Briefly, kernels
were ground under liquid nitrogen in a pre-chilled mortar, and
metabolites were extracted with methanol (500 mg kernel tissue per
2 ml methanol) containing phenyl-b-D-glucopyranoside (130 mg
ml21) as an internal standard for normalization. After 16 h
incubation, samples were centrifuged, and part of the supernatant
(150 ml) was transferred to a 2 ml autosampler vial and dried by
nitrogen at ambient temperature. Dried extracts were resuspended in
100 ml trimethylsilylimidazole/trimethylchlorosilane (TMS; 100 : 1,
v/v), and incubated at 37 uC for 1 h. The TMS products were
partitioned into the organic phase after the addition of 100 ml
isooctane and 200 ml water. Of the organic phase, 1 ml was analysed
with a GC-2010 gas chromatograph (Shimadzu) equipped with a
ZB-5 column (30 m60.25 mm, Phenomenex) and a flame-ionization detector (FID) set at 340 uC. To separate metabolites, the oven
temperature was held constant at 120 uC for 5 min, increased to
300 uC by 4 uC per 1 min, and then held constant at 300 uC for
15 min. GC-FID data were normalized to the internal standard by
multiplying each metabolite peak area by a normalization factor. To
generate the normalization factor, the largest internal standard peak
area was divided by the internal standard peak area for each
fingerprint. To minimize differences due to slight variation in
starting material, peak areas from each fingerprint were divided by
the tissue weight used for extraction. Finally, to normalize each
metabolite to fungal growth, peak areas from each fingerprint were
divided by ergosterol peak area of the sample. Arabitol, mannitol
and trehalose were quantified based on comparison with standard
curves derived from analytical standards (respectively: Alfa Aesar,
Fisher Scientific and TCI America).
RESULTS
Identification and disruption of HXK1
The genome of F. verticillioides is predicted to contain at least
six genes encoding putative hexokinases: FVEG_00957,
FVEG_10632, FVEG_06423, FVEG_02067, FVEG_01889 and
FVEG_05775 (Broad Institute; http://www.broadinstitute.
org). Conceptual translation of these genes indicated a
similar domain architecture, consisting of two characteristic
hexokinase domains: hexokinase 1 (Pfam accession no.
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Role of HXK1 in pathogenesis of F. verticillioides
PF00349) and hexokinase 2 (Pfam accession no. PF03727; Fig.
1a). A phylogenetic analysis of the putative hexokinase
proteins by neighbour-joining methods revealed three distinct
groups, each containing two proteins that shared substantial
similarity (Fig. 1a and Supplementary Fig. S1). An additional
phylogenetic analysis of hexokinases, non-catalytic hexokinases and glucokinases from F. verticillioides and selected other
fungi indicated that FVEG_00957 and FVEG_10632 group
with hexokinases, FVEG_01889 and FVEG_05775 are most
similar to other fungal glucokinases, and FVEG_02067 and
FVEG_06423 group with non-catalytic or regulatory hexokinase-like proteins (Supplementary Fig. S1). Gene sequence
FVEG_00957 (GenBank accession no. EU247513), which was
designated previously as HXK1 (Bluhm et al., 2008), was
selected for characterization based on its expression profile
during kernel colonization (Bluhm et al., 2008) and the
similarity of the predicted Hxk1 protein to a functionally
characterized hexokinase from Kluyveromyces lactis (KlHxk1,
GenBank accession no. CAA08922.1; Kuettner et al., 2010).
HXK1 has an ORF of 2329 bp with five introns, and is
predicted to encode a protein of 493 aa containing two
structurally similar domains. Ribbon diagrams depicting the
tertiary structure of KlHxk1 and the predicted tertiary
structure of Hxk1 indicated a high degree of structural
similarity (Fig. 1b). In addition, the amino acid sequence of
Hxk1 shares 71.8 % identity with the characterized hexokinase
HxkA of Aspergillus nidulans (GenBank accession no.
XP_680728.1) and 80.1 % identity with a hexokinase in
Botrytis cinerea (GenBank accession no. ABM30191.1). The
other F. verticillioides gene (FVEG_10632) that grouped with
Hxk1 in the phylogenetic analyses (Fig. 1a and Supplementary
Fig. S1) shared substantially less identity with KlHxk1 and
HxkA than Hxk1 (46 and 52.4 %, respectively), which suggests
that the protein has a substantially different tertiary structure
or phosphorylates other substrates.
To functionally characterize HXK1, the gene was deleted in
the wild-type strain via homologous recombination (Fig.
2a). We obtained 32 transformants that were resistant to
hygromycin, and these transformants were screened by
PCR with primers HXK1-1 and HYG3 (data not shown) to
identify putative deletion mutants. Two individual HXK1
deletion mutants were obtained and confirmed by
Southern analysis (Fig. 2b), and one of these mutants
was chosen for further study. To complement the mutant,
the HXK1 gene was amplified via PCR from the wild-type
Fig. 1. Phylogenetic and comparative analyses of hexokinases in F. verticillioides. (a) Neighbour-joining tree inferred from
putative hexokinase proteins of F. verticillioides. Conceptual translation of HXK1 reveals a protein predicted to contain the two
functional domains found in known hexokinase proteins (hexokinase 1 and hexokinase 2, represented by shaded boxes).
(b) Comparison of the structural model of Hxk1 of F. verticillioides with the crystal structure of KlHxk1 of K. lactis.
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Fig. 2. Disruption of HXK1 in F. verticillioides.
(a) To disrupt HXK1, a hygromycin-resistance
cassette (HYGR) was inserted into the HXK1
locus by homologous recombination. (b) For
Southern analysis (left panel), genomic DNA
was digested with XhoI (X) and probed with a
600 bp fragment (P) shown in (a). For the
complementation strain, PCR (right panel) was
performed with primers specific to the deleted
region of the HXK1 locus, and the resulting
band indicates the presence of the internal
fragment (623 bp) of HXK1. M, 1 kb size
marker. (c) Wild-type, Dhxk1 and Dhxk1-comp
were grown on various media: YEP, and DL
containing either 2 % glucose or 2 % fructose
as the sole carbon source.
strain and transformed directly into the Dhxk1 mutant. A
complemented strain (Dhxk1-comp) containing the wildtype copy of HXK1 was confirmed by PCR with primers
specific to HXK1 (Fig. 2b).
HXK1 is required for utilization of fructose
The specific substrate(s) of a hexokinase cannot be
determined conclusively from conceptual translation and
homology-based analyses of the putative protein sequence.
To evaluate substrate specificity for Hxk1, we compared
growth and sugar uptake of the Dhxk1 mutant with those
of the wild-type and Dhxk1-comp. On a medium containing fructose as the sole carbon source, the Dhxk1 mutant
was unable to grow (Fig. 2c). Complementation of the
Dhxk1 mutant restored growth on fructose, albeit not
completely compared with the wild-type strain (Fig. 2c).
However, the Dhxk1 mutant was able to grow on a medium
containing glucose as the sole carbon source (Fig. 2c).
These results indicated that HXK1 is required specifically
for utilization of fructose, and suggest that other putative
hexokinases are unable to compensate for Hxk1 function in
the presence of fructose, despite moderate to substantial
sequence similarity (Fig. 1a).
Phosphorylation of a hexose by a hexokinase prevents
diffusion out of the cell and commits the hexose molecule
to a specific cellular process, i.e. glycolysis (Romano, 1982).
To determine whether HXK1 is required for fructose
uptake and/or retention, the amount of free sugar was
quantified in resuspension cultures of the wild-type,
mutant and complemented strain. The Dhxk1 mutant was
unable to take up and/or retain fructose, as evidenced by
the relatively constant amount of soluble fructose present
in the resuspension medium; in comparison, a 50 %
depletion of fructose was observed in resuspension cultures
of the wild-type or complemented strain (Fig. 3).
Furthermore, dry weight measurements indicated that
Dhxk1 did not grow in DL medium containing fructose,
whereas both the wild-type and Dhxk1-comp grew
considerably (Table 1). In contrast, the three strains had
similar growth in DL medium containing glucose as the
sole carbon source (Table 1). Together, these results
Fig. 3. Sugar analysis. All strains were grown
on YEP medium for 3 days. Mycelia were
transferred to defined medium containing
either 2 % fructose or 2 % glucose as the sole
carbon source. At each time point, sugars that
remained in the medium were measured in
three biological replicates. The experiment was
performed twice with similar results (data from
a representative experiment are shown).
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Role of HXK1 in pathogenesis of F. verticillioides
Table 1. Growth of the wild-type, Dhxk1 mutant and complemented strain on maize kernels and liquid media
Strain
Wild-type
Dhxk1
Dhxk1-comp
Maize kernel medium*
DL mediumD
Whole kernel
Endosperm tissue
Germ tissue
DL-glucose
DL-fructose
744±53
482±59
742±42
566±6
207±2
550±15
640±69
702±91
674±78
0.37 (+0.07)
0.34 (+0.04)
0.37 (+0.07)
0.38 (+0.16)
0.22 (+0)
0.38 (+0.16)
*Growth on maize kernel medium is represented by ergosterol concentration (values show mean±SD of three replicates). Ergosterol values are mg
ergosterol (g maize kernels)21.
DAfter transferring mycelia grown on YEP medium, growth as dry mycelial weight (g) was measured at 65 h (DL-glucose) or 96 h (DL-fructose).
Values in parentheses indicate the increase in weight compared with the initial inoculum. Similar results were obtained in two independent
experiments.
indicated that HXK1 is involved primarily in the uptake
and/or utilization of fructose.
Deletion of HXK1 impairs conidiation and alters
hyphal morphology
To identify roles for HXK1 in processes other than primary
metabolism, the impact of HXK1 disruption on fungal
development was examined. The Dhxk1 mutant produced
fewer conidia than the wild-type or the Dhxk1-comp strain
when provided with glucose, sucrose or maltotriose as the
sole carbon source (Fig. 4a). Also, the mutant grew more
slowly than the wild-type or complemented strain when
provided with the aforementioned carbon sources (Fig. 4a).
Furthermore, when grown on YEP+fructose medium, the
mutant produced swollen hyphae and conidia, although the
wild-type grew normally (Fig. 4b). Interestingly, when
grown on YEP+glucose medium, the mutant was morphologically indistinguishable from the wild-type (Fig. 4b),
which suggested a specific response to fructose.
HXK1 is required for infection and colonization of
maize kernels, but not stalks
Observations that the Dhxk1 mutant was impaired in
growth when provided with specific sugars or carbohydrates (Fig. 4) gave rise to the question of whether the
mutant would be reduced in virulence during infection of
Fig. 4. Effects of HXK1 on conidiation and
fungal development. (a) Conidiation and radial
growth were measured in the wild-type (white
bars) and strain Dhxk1 (black bars) grown on
defined medium containing glucose, fructose,
sucrose or maltotriose as the sole carbon
source. To evaluate conidiation, 0.5 cm diameter agar plugs were taken at 5 days after
inoculation and resuspended in 1 ml water.
Results are mean±SD of three biological
replicates. Radial growth was measured from
3 day-old cultures. (b) Hyphal swelling was
observed in strain Dhxk1. The wild-type and
the mutant strain were grown on YEP+
glucose and YEP+fructose for 3 days. White
arrows indicate swelling observed in the
mutant grown on YEP+fructose.
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distinct maize tissues, e.g. kernels versus stalks. To evaluate
virulence on maize kernels, ears of inbred B73 were
inoculated with the wild-type, Dhxk1 mutant or Dhxk1comp strain in greenhouse assays. At 7 days after
inoculation, the wild-type and the complemented strain
aggressively colonized maize kernels, whereas growth of the
Dhxk1 mutant was restricted to wound sites (Fig. 5a).
Dissection of colonized kernels revealed that the endosperm of kernels inoculated with Dhxk1 remained yellow
and firm, but the endosperm tissues inoculated with the
wild-type and complemented strain were completely
colonized (data not shown). Additionally, on autoclaved
cracked whole-kernel and endosperm tissue media, Dhxk1
produced 64 and 36 %, respectively, of the ergosterol
produced by the wild-type (Table 1); ergosterol is
commonly used as a quantitative measure of fungal
growth. However, when inoculated on germs excised from
mature kernels, growth of the mutant was similar to that of
the wild-type and Dhxk1-comp strain (Table 1). These
results indicated that Dhxk1 was impaired in its ability to
utilize carbohydrates found specifically in maize endosperm tissues.
To evaluate virulence on maize stalks, a commercial hybrid
was inoculated with the wild-type, Dhxk1, and Dhxk1comp in greenhouse assays. Somewhat surprisingly, the
wild-type, Dhxk1, and Dhxk1-comp were consistently
indistinguishable (Fig. 5b), which indicates that HXK1
does not play an important role during colonization of
stalk tissue.
HXK1 is required for wild-type levels of FB1
biosynthesis during kernel colonization
To determine whether HXK1 affects fumonisin biosynthesis, we measured FB1 production by the wild-type, the
Dhxk1 strain and the Dhxk1-comp strain during growth on
cracked maize kernel medium. FB1 levels were normalized
to fungal growth [ergosterol content (g maize)21]. The
mutant produced fivefold less FB1 than the wild-type, and
production of FB1 was restored in the complemented strain
(Fig. 6, Supplementary Table S1). These results showed
that the reduction in FB1 biosynthesis was fully attributable
to disruption of HXK1.
Metabolic profiling reveals that deletion of HXK1
impairs trehalose biosynthesis and utilization of
hexose sugars during colonization of maize
kernels
Because of the growth impairment of Dhxk1 on maize
kernels, metabolic profiling was performed to determine
whether disruption of HXK1 incurred global changes
throughout the metabolome during kernel colonization.
To this end, maize kernels were inoculated with the wildtype, the Dhxk1 strain and the Dhxk1-comp strain, and
metabolic profiles of polar metabolites such as carbohydrates were obtained. A total of 22 metabolites were
detected in the colonized maize kernels (Supplementary
Table S2). Comparisons of relative, normalized concentrations of each metabolite indicated that disruption of HXK1
did not cause widespread changes in the metabolome
(Supplementary Fig. S2). However, consistent with a role
for HXK1 in sugar uptake and/or retention, disruption of
HXK1 significantly increased the amount of sucrose,
fructose and glucose present in colonized kernels compared
with the wild-type or Dhxk1-comp strain (Supplementary
Table S2). Presumably, this difference resulted from the
rapid catabolism of the sugars during growth and
colonization of the wild-type and the complemented
strain. Additionally, disruption of HXK1 consistently
reduced the concentration of a metabolite eluting at
36.448 min (Fig. 7), which has previously been identified
by MS as trehalose (Smith et al., 2011). This identification was confirmed by spiking experiments and
Fig. 5. Effect of HXK1 on pathogenesis. (a) Ear rot assay. Conidia (106 ml”1) of the wild-type, Dhxk1 or Dhxk1-comp were
inoculated into maize kernels with a needle. Pictures were taken 7 days after inoculation. (b) Stalk rot assay. Maize stalks
(internodal regions) were inoculated with 105 spores of fungal strains and incubated in a greenhouse. After 5 days, the maize
stalks were split longitudinally to assess stalk rot severity. A maize stalk inoculated with sterile water is shown as the negative
control.
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Microbiology 157
Role of HXK1 in pathogenesis of F. verticillioides
Fig. 6. Effect of HXK1 on FB1 biosynthesis. FB1 biosynthesis was
normalized to growth with the following equation: [FB1 production
(mg g”1)/ergosterol contents (mg g”1)]¾100. For the analysis, all
strains were grown on mature cracked kernels for 7 days, and FB1
and ergosterol were extracted from ground cultures. The values
were generated by three biological replicates, and two independent experiments showed comparable results.
chromatographic analyses of a commercially available
standard (data not shown).
Among filamentous fungi, hexokinases have been postulated to play important roles in the biosynthetic pathways
of trehalose and sugar alcohols, both of which are
important in mitigating environmental stresses such as
heat and oxidative shock (Fillinger et al., 2001). Our
analysis indicated that arabitol and mannitol levels were
similar in the wild-type and the Dhxk1 strain (Fig. 7,
Supplementary Table S2). However, accumulation of
trehalose in kernels colonized by Dhxk1 was twofold less
than that of kernels colonized by either the wild-type or the
complemented strain (Fig. 7), whereas in uninoculated
kernels, trehalose was not detected (Supplementary Fig.
S2). Additionally, the Dhxk1 strain produced 30 % less
trehalose than the wild-type during colonization of
autoclaved maize kernels (data not shown), and 35 % less
trehalose than the wild-type when grown on YEP liquid
medium containing 10 % glucose (Supplementary Fig. S3).
Intriguingly, levels of glucose 6-phosphate were comparable between the mutant and the wild-type (Supplementary
Fig. S3) during growth on YEP liquid medium containing
10 % glucose, which suggests that the reduction in
trehalose biosynthesis observed in the Dhxk1 strain may
be related to carbohydrate sensing rather than the ability to
synthesize glucose 6-phosphate, a precursor of trehalose
biosynthesis. Taken together, these results showed that
trehalose biosynthesis is consistently reduced by disruption
of HXK1 across a range of environmental conditions.
http://mic.sgmjournals.org
Fig. 7. Quantification of arabitol, mannitol and trehalose in fungal
cultures. White bars, wild-type; black bars, Dhxk1; grey bars,
Dhxk1-comp. Inoculated kernels obtained from ear rot assays were
selected randomly for analysis of fungal metabolites. The values
were normalized with respect to ergosterol and an internal
standard, and analyses were performed with three biological
replicates. The significance of differences among means was
calculated with Tukey’s honest significance test, and significant
differences (P,0.05) are indicated with an asterisk.
Deletion of HXK1 reduces osmotic stress
tolerance
The observation that trehalose biosynthesis was reduced in
the Dhxk1 mutant gave rise to the question of whether or
not the mutant displayed increased sensitivity to environmental stresses. In many fungi, trehalose is essential for
virulence and tolerance of environmental stresses, including heat, cold, starvation, desiccation, and osmotic and
oxidative damage (Gadd et al., 1987; Lewis et al., 1995;
Ngamskulrungroj et al., 2009; Thevelein, 1984). To
investigate osmotic stress tolerance in the Dhxk1 mutant,
we compared growth (colony diameter) of the wild-type
and Dhxk1 mutant on YEP media containing glucose,
fructose or glycerol in the presence or absence of 1 M NaCl
(Fig. 8). In the presence of 1 M NaCl, growth of the mutant
was approximately 65 % less than that of the wild-type
when fructose was the sole carbon source (Fig. 8a, b), but
when glucose or glycerol was supplied to the YEP medium,
the mutant exhibited approximately 10 % reduced growth
compared with the wild-type (Fig. 8b). In the absence of
NaCl, the reduced growth rate of the mutant was similar
on all YEP media, regardless of carbon source (Fig. 8). We
confirmed that the complemented strain restored the
growth defect on YEP+fructose medium in the presence
of 1 M NaCl (data not shown). Additionally, tolerance of
oxidative stress was evaluated by measuring growth on YEP
medium (containing glucose or fructose as the sole carbon
source) supplemented with H2O2 (10 mM) and Congo red
(50 mg ml21). Under these conditions, growth rates of the
mutant and wild-type were similar, regardless of carbon
source (data not shown).
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H. Kim and others
Fig. 8. Salt stress sensitivity. (a) Growth of wild-type and Dhxk1 on YEP+fructose in the presence or absence of 1 M NaCl.
Pictures were taken 3 days after point inoculation [2 ml of conidia suspension (106 ml”1)]. (b) Colony diameters of the wild-type
(white bars) and Dhxk1 (black bars) were measured on YEP medium with glucose (Glc), fructose (Fru) or glycerol (Glyc) and
with or without NaCl. All SDs for three replicates were ,0.1 cm.
DISCUSSION
The molecular events underlying fungal responses to sugar,
particularly glucose, are understood best in the yeast
Saccharomyces cerevisiae (Santangelo, 2006). Three components are known to be involved in the sensing of glucose
and the initiation of a molecular response in yeast: glucose
sensors (Snf3p and Rgt2p), hexokinase (Hxk2p) and the
G-protein-coupled receptor (Gpr1p) (Hohmann et al.,
1999; Kraakman et al., 1999; Ozcan et al., 1998).
Filamentous fungi also have mechanisms to perceive and
respond to sugar, some of which are unique to higher
fungi, and some of which are similar to those found in
yeast. Evidence from A. nidulans indicates that a system
involving heterotrimeric G-proteins coupled with adenylate cyclase is the major mechanism for glucose sensing and
the subsequent changes in the transcriptome (Lafon et al.,
2005). Also, two kinases that phosphorylate hexoses have
been identified in A. nidulans (GlkA and HxkA) and B.
cinerea (Glk1 and Hxk1). However, these enzymes are not
involved in carbon catabolite repression, which has a
different regulatory mechanism from that of yeast (Flipphi
et al., 2003; Rui & Hahn, 2007). Hexokinases in fungi and
plants are known to be involved in signalling networks for
controlling growth and development in response to the
changing environment (Fleck & Brock, 2010; Moore et al.,
2003).
Beyond the crucial role that hexokinase plays in glycolysis,
the regulatory functions of hexokinases, including
responding to and signal transduction by sugars, are
poorly understood in filamentous fungi. Rui & Hahn
(2007) have shown that a B. cinerea mutant disrupted in
HXK1 fails to grow on fructose, and grows poorly on
media containing a variety of other carbon sources. In
addition, the mutant is virulent on tomato leaves, but
produces small lesions and reduced conidiation on apple
fruit, which suggests that the regulation of sugar catabolism
plays a major role in pathogenicity and disease development. Furthermore, Fleck & Brock (2010) have shown
2666
that during infection of mouse lung, GLKA and HXKA of
Aspergillus fumigatus are highly expressed, suggesting the
involvement of the genes in virulence. These results are
consistent with our observation that strain Dhxk1 is
substantially impaired in pathogenicity during infection
of maize. Moreover, our results indicating that HXK1 is
involved in ear rot but not stalk rot suggest that F.
verticillioides possesses distinct, carbohydrate-dependent
mechanisms for pathogenicity that depend heavily on the
types and amount of carbon sources present in a particular
plant tissue.
Our observations that HXK1 is required for FB1 biosynthesis establish an important new genetic link between
primary and secondary metabolism in F. verticillioides.
Keller & Sullivan (1996) reported that glucose did not
repress FB1 production in Fusarium proliferatum, a close
relative of F. verticillioides, when glucose concentrations
were maintained between 30 and 40 g l21 at pH 3.5 in a
bioreactor. These results suggest that genes involved in
carbon catabolite repression do not regulate fumonisin
biosynthesis, which is consistent with the lack of consensus
binding sites for CreA in the promoters of FUM1 and
FUM21. However, in F. verticillioides, CreA may indirectly
regulate fumonisin biosynthesis through the regulation of
sugar transporters and sugar kinases (Bhatnagar et al.,
2003). This hypothesis is supported by our observation that
HXK1 has four putative CreA binding sites (2356, 2245,
242 and 224 bp) within 500 bp upstream of the start
codon. However, the exact mechanism through which
HXK1 affects FB1 production remains to be determined
and will require further genetic dissection.
In fungi, trehalose and polyols such as mannitol serve as
protectants against environmental stress. For example,
conidia produced by an A. nidulans mutant disrupted in
TPSA, which encodes a trehalose-6-phosphate synthase
catalysing the first step in trehalose biosynthesis, show a
rapid loss of viability when subjected to heat and oxidative
stress, whereas conidia of the wild-type retain viability
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Microbiology 157
Role of HXK1 in pathogenesis of F. verticillioides
under the same treatments (Fillinger et al., 2001). Hounsa
et al. (1998) showed that in S. cerevisiae, mutant strains
(Dtps1Dtps2 and Dtps1Dhxk2) are unable to produce
trehalose and are also more sensitive to osmotic stress.
Additionally, disruption mutants in TPS1 of A. nidulans
and B. cinerea are more sensitive to osmotic stress and heat
stress, respectively, than the wild-type strains (Doehlemann
et al., 2006; Fillinger et al., 2001). Trehalose biosynthesis is
catalysed by the action of trehalose-6-phosphate synthase
and trehalose-6-phosphate phosphatase from glucose
6-phosphate. However, our observation that Dhxk1 was
not able to take up fructose or grow on fructose as a sole
carbon source gave rise to a question: why does Hxk1 have
an effect on trehalose biosynthesis, but not on mannitol
biosynthesis, considering that mannitol is produced by the
action of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphate phosphatase from fructose 6-phosphate?
We cannot exclude the possibility that Hxk1 of
F. verticillioides is directly or indirectly involved in the
phosphorylation of glucose; this possibility is supported by
our observations of reduced growth of the mutant on DL
medium containing glucose. Furthermore, we observed
that mannitol accumulation was increased up to threefold
in the Dhxk1 strain compared with the wild-type when
grown on autoclaved maize kernels (data not shown).
These results suggest that disruption of HXK1 in
F. verticillioides has a conditional effect on mannitol
biosynthesis, although the underlying mechanism is not
clear at this time.
In some fungi, trehalose has undetermined functions in
pathogenesis. For example, trehalose metabolism regulates
development and pathogenicity in Magnaporthe oryzae; a
deletion mutant of TPS1, encoding trehalose-6-phosphate
synthase, fails to synthesize trehalose, sporulates poorly, and
is reduced in pathogenicity (Foster et al., 2003). Also, the
human fungal pathogen Cryptococcus sp. requires trehalose
biosynthesis for virulence (Ngamskulrungroj et al., 2009).
Besides the role of trehalose in pathogenicity and fungal
development, in yeast, trehalose 6-phosphate has been
postulated to inhibit Hxk2p (Thevelein & Hohmann, 1995).
As a consequence of this inhibitory effect, trehalose
6-phosphate levels can affect the flow of glucose into
glycolysis with an overall effect of regulating sugar
metabolism. Recently, trehalose-6-phosphate synthase in
M. oryzae was shown to affect the pentose phosphate
pathway and virulence, but did not affect hexose kinase
activity (Wilson et al., 2007). The authors proposed a model
in which trehalose-6-phosphate synthase has a central role
that affects glucose catabolism, pathogenicity, nitrogen
metabolism, and conidiation. An interesting observation
made by those authors with relevance to our findings in
F. verticillioides is that trehalose levels are reduced in a Dhxk1
mutant. The exact relationship between reduced trehalose
biosynthesis and reduced virulence in F. verticillioides is
unclear, however, and warrants further study.
In summary, HXK1 represents a critical genetic link
between primary metabolism, pathogenesis and secondary
http://mic.sgmjournals.org
metabolism in F. verticillioides. Characterization of HXK1
creates new insights into how F. verticillioides senses
distinct tissues of maize during pathogenesis, and also
provides tools for further molecular dissection of the
complex signalling pathways governing pathogenesis and
mycotoxigenesis in F. verticillioides.
ACKNOWLEDGEMENTS
We thank Ms Shantae Wilson for technical assistance. Funding for
this research was provided by the Arkansas Center for Plant Powered
Production (P3) and the Arkansas Biosciences Institute. Funding was
also provided by USDA/NIFA/AFRI, award number 10-65108-20567,
to C. P. W.
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