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Contents lists available at ScienceDirect
Fungal Genetics and Biology
journal homepage: www.elsevier.com/locate/yfgbi
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Regular Articles
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Analysis of cytochrome b5 reductase-mediated metabolism in the
phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities
implicated in virulence
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Mark C. Derbyshire a,⇑, Louise Michaelson b, Josie Parker c, Steven Kelly c, Urvashi Thacker d,
Stephen J. Powers e, Andy Bailey f, Kim Hammond-Kosack a, Mikael Courbot g, Jason Rudd a,⇑
a
Department of Plant Biology and Crop Science, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, UK
Department of Biological Chemistry and Crop Protection, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, UK
c
Centre for Cytochrome P450 Diversity, Institute of Life Science, College of Medicine, Swansea University Singleton Park, Swansea SA2 8PP, Wales, UK
d
Syngenta, Jealott’s Hill, Bracknell, Berkshire RG42 6EY, UK
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Department of Computational and Systems Biology, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, UK
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Bristol University, Senate House, Tyndall Avenue, Bristol BS8 1TH, UK
g
Syngenta, Syngenta AG, Schaffhauserstrasse, CH-4332 Stein, Switzerland
b
a r t i c l e
i n f o
Article history:
Received 30 December 2014
Revised 19 May 2015
Accepted 20 May 2015
Available online xxxx
Keywords:
Septoria tritici
Mycosphaerella graminicola
Dimorphic fungi
Fatty acids
Cytochrome P450
CYP51
a b s t r a c t
Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural
fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron
transfer enzymes because these are often important for cell function. Therefore characterisation of genes
encoding such enzymes may be important for the development of novel disease intervention strategies.
Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which
in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids
and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal
CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore DZtCBR1 mutant strains were generated through targeted gene disruption. These
strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation.
DZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC–MS and
HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron
donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study
reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon
fungal sterol biosynthesis.
Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
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Abbreviations: CBR, cytochrome b5 reductase; b5, cytochrome b5; CPR,
cytochrome P450 reductase; CYP, cytochrome P450; STB, Septoria tritici blotch;
DPI, days post inoculation; WT, wild-type; HPLC, high pressure liquid chromatography; GC–MS, gas chromatography–mass spectrometry; FAME, fatty acid methyl
ester; FPKM, fragments per kilobase per million mapped fragments; RNA-seq, RNA
sequencing; LCB, sphingolipid long chain base.
⇑ Corresponding authors at: Centre for Crop and Disease Management, Curtin
University, Kent Street, Bentley, WA 6102, Perth, Australia. Tel.: +44 7957941621
(M.C. Derbyshire). Tel.: +44 (0) 1582 763 133x2187 (J. Rudd).
markcharder@gmail.com
(M.C.
Derbyshire),
E-mail
addresses:
jason.rudd@rothamsted.ac.uk (J. Rudd).
1. Introduction
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Septoria tritici blotch (STB) caused by the wheat leaf-specific
Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. The most significant
‘within field’ damage caused by Z. tritici is mediated through asexual spores. These spores are rain splash propagated throughout the
wheat canopy where they attach to leaf surfaces and then germinate into infectious hyphae which penetrate the plant through
stomata. This takes place within 24 h of spores landing on the leaf’s
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http://dx.doi.org/10.1016/j.fgb.2015.05.008
1087-1845/Ó 2015 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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M.C. Derbyshire et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx
surface and is followed by a symptomless phase of slow intercellular colonisation within the leaf lasting approximately 10 days
(Orton et al., 2011). Following this initial symptomless period the
fungus elicits a rapid onset of host cell necrosis, which bears hallmarks of plant programmed cell death. At this point Z. tritici
switches to a necrotrophic mode of feeding and begins accumulating biomass rapidly, generating asexual fruiting bodies (termed
pycnidia) within the developing necrotic lesions (Keon et al.,
2007). Following infection of a susceptible wheat cultivar by a
wild-type (WT) strain the complete disease cycle takes approximately 21 days, culminating in the development of mature pycnidia on infected leaves. The masses of asexual spores produced by
the pycnidia (pycnidiospores) are multicellular units most frequently composed of four to six cells (Eyal et al., 1987).
There is currently a major deficit in commercially relevant
STB-resistant germplasm. As a result Z. tritici is a major target for
fungicides, especially in temperate regions where it is most prevalent: approximately 70% of fungicides (equating to a cost of
>€400 m) sold in the EU are used to prevent STB (O’Driscoll et al.,
2014). Due in part to the intense selective pressure caused by the
widespread use of just a handful of antifungal chemistries, fungicide resistance in Z. tritici is a major problem (Cools and Fraaije,
2008; Siah et al., 2014; Taher et al., 2014). Many fungicides target
enzymes involved in electron transfer systems as such systems are
often essential for cell function. Therefore, analysis of genes encoding hitherto uncharacterised electron transfer enzymes in Z. tritici
may be relevant for future development of novel antifungal
chemistries.
Aside from those present on the chloroplastic and mitochondrial membranes, the two major eukaryotic electron transfer systems are the cytochrome P450 reductase (CPR)-dependent and
microsomal cytochrome b5 reductase (CBR)-dependent pathways.
The former provides the electrons necessary for the function of
cytochrome P450 (CYP) enzymes, which are involved in various
biological processes such as detoxification of xenobiotic compounds and biosynthesis and metabolism of lipids and secondary
metabolites (George et al., 1998; Mutch et al., 2007; Lepesheva
and Waterman, 2007; Richter et al., 2008). The latter usually
involves transfer of electrons through CBR then cytochrome b5
(b5) to terminal electron acceptor desaturase or hydroxylase
enzymes. The major functions of the desaturases and hydroxylases
in this system are to catalyse double bond formation between carbon atoms and addition of hydroxyl groups during the biosynthesis
of unsaturated fatty acids (UFAs) and the more complex lipids the
sphingolipids and sterols (Huang et al., 1999; Knutzon et al., 1998;
Michaelson et al., 2013; Grinstead and Gaylor, 1982; Moreno-Perez
et al., 2011). In addition to its major role in electron transfer to
desaturases and hydroxylases, cytochrome b5 is also known to be
involved in electron transfer to some CYP enzymes (Henderson
et al., 2013; Gan et al., 2009), though the functional relationship
between CYPs and the CBR-b5 pathway remains largely elusive.
In addition to the microsomal CBR-b5 system there is also a mitochondrial CBR-b5 system (Hahne et al., 1994). Though the function
of this system also remains largely elusive, it has been linked with
metabolism of xenobiotics and lipid biosynthesis (Nikiforova et al.,
2014; Neve et al., 2012; Glory and Thiruvenangdam, 2011).
To date studies on microsomal CBR enzymes have been limited
to the model yeast Saccharomyces cerevisiae, the industrial arachidonic
acid-producing
fungus
Mortierella
alpina
(Class:
Zygomycota), the model white-rot fungus Phanerochaete
chrysosporium (Order: Basidiomycota), and the filamentous fungus
Mucor racemosus (Class: Zygomycota). In S. cerevisiae it was
demonstrated that the sole microsomal CBR present in the fully
sequenced genome is able to provide the reducing power necessary
for the function of a CYP enzyme, CYP51, needed for biosynthesis of
the main fungal sterol, ergosterol, (albeit in reconstituted system)
rendering CPR dispensable (Lamb et al., 1999; Sutter and Loper,
1989). Similarly, studies in P. chrysosporium showed that the
CBR-b5 system is able to efficiently provide electrons to the
enzyme CYP63A2, a multifunctional CYP (Syed et al., 2011).
These findings offered some insight into the potential overlap in
function between the CPR and CBR systems in fungi. In M. alpina
two CBRs were identified and cloned in the late 1990s. The first
of these was heterologously expressed in Aspergillus oryzae leading
to an increase in ferricyanide reduction activity (Sakuradani et al.,
1999), though no analyses of the in situ function of this enzyme
were carried out. The second of these was used in conjunction with
the first in a phylogenetic analysis, which demonstrated evolutionary divergence of mammalian CBR enzymes from those of fungi
and plants (Certik et al., 1999). In M. racemosus, CBR was cloned
and expressed in E. coli (Mirzaei et al., 2010), though again no analyses of the biological function of the enzyme in the host organism
were conducted.
Whilst much research has been carried out on mammalian
(Celik et al., 2013; Elahian et al., 2014) and plant CBRs (Wayne
et al., 2013; Kumar et al., 2006; Shockey et al., 2005; Bagnaresi
et al., 2000), there have been no further investigations into the
roles of these enzymes in fungi. In order to address this, we have
analysed members of the CBR gene family in Z. tritici which is a
plant pathogen in the Dothideomycete class of fungi. In the last
decade the molecular interaction between this fungus and its host
has come under greater scrutiny (Gohari et al., 2014; Lee et al.,
2014; Suffert et al., 2013; do Amaral et al., 2012; Motteram et al.,
2009; Marshall et al., 2011). However, there have been little in
the way of investigations into the metabolic processes important
for growth and plant pathogenesis. Based upon analysis of the fully
sequenced genome of the Z. tritici reference isolate IPO323
(Goodwin et al., 2011), and recent RNA sequencing (RNA-seq) data
Rudd et al., 2015 we determined that only one of the three putative
CBRs in Z. tritici, ZtCBR1, was highly expressed both in vitro and
throughout plant infection. By generating targeted ZtCBR1 disruption strains it was shown that this gene is essential for full virulence in wheat. In vitro observation of DZtCBR1 strains revealed
various morphological and biochemical defects including reduced
spore size, reduced filamentous growth and almost a complete lack
of asexual sporulation at the end of the infection cycle in planta.
Perturbations in sphingolipid, sterol and fatty acid biosynthesis
pathways were identified using GC–MS and HPLC analyses in the
DZtCBR1 strain. This study represents the first functional analysis
of members of the CBR gene family in a plant pathogenic
Ascomycete fungus, and highlights several CBR1-regulated functions that underpin virulence in Z. tritici.
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2. Materials and methods
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2.1. Identification of sequence homologues of yeast cytochrome b5
reductase (ScCBR1) in Z. tritici and other filamentous fungi
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In order to identify putative microsomal CBR genes in several
unrelated Ascomycete and Basidiomycete fungi including Z. tritici, BLASTp analyses were carried out via the NCBI BLASTp suite
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_
TYPE=BlastSearch&LINK_LOC=blasthome) using the S. cerevisiae
CBR sequence, ScCBR1 (GenBank accession: CAA82214.1), as a
query (fungal genomes queried are detailed in Supplementary
Table 2).
BLAST hits above 30% amino acid (aa) identity with an e value
lower than e 10 were retrieved and subjected to PFam domain prediction (Finn et al., 2014) to determine whether they contained flavin adenine dinucleotide (FAD)-binding (PFam identifier: PF00667)
and nicotinamide adenine dinucleotide (NAD)-binding domains
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Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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(PFam identifier: PF08030), which are both universally present in
CBRs.
Sequences
that
contained
additional
molybdopterin-binding domains (PFam identifier: PF00174) were
excluded from further analyses as this is a feature common to
nitrate reductases, which are structurally closely related to CBRs
but involved in different processes (Truong et al., 1991).
The putative microsomal CBR sequences were then subjected to
a TargetP analysis (Emanuelsson et al., 2000) (accessed via: http://
www.cbs.dtu.dk/services/TargetP/) to determine whether the predicted proteins were potentially localised to mitochondria or the
endoplasmic reticulum. The S. cerevisiae mitochondrial CBR
sequence, ScMCR1 (GenBank accession: NP_012221.2), was then
used as a query in further BLASTp analyses to determine whether
CBRs identified with a TargetP prediction of localisation to mitochondria were indeed more closely related to this sequence than
SsCBR1. This step was needed to differentiate between sequence
homologues as mitochondrial CBRs are highly similar to microsomal CBR sequences owing to the presence of the highly conserved
FAD- and NAD-binding domains necessary for their functions.
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2.2. In vitro culture conditions and fungal strains
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Fungal spores were routinely cultured on yeast peptone dextrose (YPD) agar for six days at 18 °C. For RNA-seq analysis spores
of the WT reference isolate IPO323 were grown in Czapek Dox
broth (CDB) minimal medium and potato dextrose broth (PDB)
nutrient-rich medium. Fungal cultures were propagated in shaking
flasks at 220 rpm and 18 °C for 3 days for PDB or 5 days for CDB
and then harvested via vacuum filtration, as detailed in Rudd
et al. (2015). These incubation periods were determined to be
within the logarithmic growth phase for Z. tritici. For analysis of
fatty acid methyl ester (FAME), sphingolipid long chain base
(LCB) and sterol content the DKu70 strain (treated as WT) Bowler
et al., 2010 and the DZtCBR1-1 strain generated in this study were
propagated under the same conditions in yeast peptone dextrose
(YPD) liquid medium. Spores were harvested via vacuum filtration
after 4 days of growth and snap-frozen in liquid nitrogen. Prior to
analysis of sterol content, spores were freeze-dried. The same culture conditions were used to grow spores for microscopy. To
induce filamentous growth in vitro, spores were spot-inoculated
onto 1% agar from a spore suspension of 1 106 spores ml 1
according to (Motteram et al., 2011).
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2.3. Growth and inoculation of plants
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Seventeen day old seedlings of the STB-susceptible wheat cultivar Riband were used for all plant infection assays and RNA-seq
analysis. Seeds were pre-germinated on wet sand at 10% relative
humidity for 3 days prior to potting and subsequently kept with
a 16 h daylight cycle. Adaxial surfaces of second leaves were inoculated according to (Keon et al., 2007) with spore suspensions at a
density of 2 106 spores ml 1 in 0.1% Silwet in sterile water. For
infection assays, mock leaves were inoculated with 0.1% Silwet
only. Plant inoculation for RNA-seq analysis conducted in the previous study (Rudd et al., 2015) followed the same procedure, without the pre-germination step, using a spore density of
106 spores ml 1 in 0.1% Tween20 in sterile water.
For analysis of asexual fungal sporulation, 8–12 replicate leaves
from independently inoculated wheat seedlings randomly distributed in a walk-in temperature, humidity and light-controlled
artificial environment were collected at either 21 or 34 days post
inoculation (DPI). For the previously published RNA-seq data, each
of two biological replicate plant samples were made up of 5 leaves
collected from independent plants randomly distributed in a single
walk-in temperature and humidity-controlled glasshouse. Samples
were collected at one, four, nine, 14 and 21 DPI. Leaves collected
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for RNA-seq were immediately frozen in liquid nitrogen,
freeze-dried, then ground to fine powder in liquid nitrogen before
RNA extraction.
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2.4. RNA extraction and RNA sequencing
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All aspects relating to RNA-seq analysis of fungal gene expression during growth in Czapek-Dox and Potato Dextrose broths
and at five time points of plant infection are described in detail
in a previous study (Rudd et al., 2015). To summarise, the following
principle procedures were followed: Total RNA was isolated from
freeze-dried tissues using the Trizol procedure (Chomczynski and
Sacchi, 1987) incorporating a final LiCl2 precipitation. All samples
(single-end) were mapped with TopHat (v2.0.6) against the Z. tritici
genome (-G Mycosphaerella_graminicola.MG2.16.gtf) (Trapnell
et al., 2012). Cufflinks (v2.1.1) was used to calculate FPKM values
for reference annotations (-G Mycosphaerella_graminicola.MG2.1
6.gtf)
but
excluding
genes
annotated
with
rRNA
(-M rRNA_genes.gtf). Differential expression analysis was done
with cuffdiff (cuffdiff -u -M rRNA_genes.gtf -b Mycosphaerella_gra
minicola.MG2.16.dna.toplevel.fa).
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2.5. Agrobacterium tumefaciens – mediated targeted disruption of
fungal genes
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In order to target ZtCBR1 for gene function ablation the plasmid
pNOV2114 was used (Motteram et al., 2009). Two sequences flanking ZtCBR1 were obtained from the JGI genome for Z. tritici (http://
genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html) and PCR amplified. Amplified sequences were then purified and inserted into
pNOV2114 either side of a hygromycin resistance cassette (hph)
(also inserted as purified PCR product) under a trpC promoter.
The same procedure was used to generate transformation vectors
for ZtCBR2 and ZtCYP-24, though the plasmid used was pCHYG
(Motteram et al., 2009), which already contained hph under the
trpC promoter. All primers and added restriction sites used for
these procedures are detailed in Supplementary Table S1 and
Supplementary Fig. S1, A shows diagrams indicating positions
and sizes of flanking sequences relative to genes of interest.
Agrobacterium tumefaciens – mediated transformation of
Z. tritici spores was carried out according to (Zwiers and De
Waard, 2001) with slight modifications. Instead of using the
antibiotic Cefotaxim, Timentin was used in transformant selection
plates as it was found to be more efficient for removal of residual
Agrobacterium tumefaciens after transformation. All transformations were carried out in a DKu70 background as disruption of this
gene has been shown to prevent ectopic insertion whilst maintaining WT growth and virulence (Bowler et al., 2010); the DKu70
strain used in transformation was treated as WT in all subsequent
experiments.
Transformant colonies were
sub-cultured
twice on
hygromycin-selective agar (50 ug ml 1). To confirm integration of
hph+trpC at the desired locus and in the correct orientation, a primer from within hph and within 200 bp of a flanking region used to
guide insertion were used to amplify a diagnostic region of approximately 1.5 kb. A single band in gDNA of mutant strains and lack
thereof in gDNA of the WT was considered representative of successful targeted disruption. All primers used for these procedures
are detailed in Supplementary Table 1. At least three disruption
strains were generated for each gene of interest (Supplementary
Fig. S1, B). Two DZtCBR1 strains were carried forward for further
analysis and arbitrarily named DZtCBR1-1 and DZtCBR1-2. Only
single DZtCBR2 and DZtCYP-24 strains were fully analysed in planta
as preliminary testing of several strains all showed no apparent
reductions in virulence.
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Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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2.6. Quantification of asexual sporulation in infected leaf tissue
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To assess the degree of asexual sporulation exhibited by Z. tritici
strains at the stated time points post inoculation, leaf samples
were kept for a further 48 h at 100% relative humidity in darkness
at 18 °C. Leaves were then submerged in 1 ml of distilled water and
left overnight. Submerged leaves were vortexed for 10 s and
spores released into the water were counted using a haemocytometer. A total of 8–12 leaves each taken from individual wheat
seedlings were analysed this way for each fungal strain tested.
Analysis of variance (ANOVA) was used on raw data for
DZtCYP-24 and DZtCBR2 and log2e -transformed data for the
DZtCBR1 strains alongside the WT to assess differences in the
amount of asexual sporulation. When a significant difference
(p < 0.05, F-test) was found, Fisher’s least significant difference
(LSD) test was used to determine significant (p < 0.05) pairwise differences between strains.
Micrographs of mutant and WT spores were generated via
bright field imaging using a Zeiss LSM 780 microscope (Carl Zeiss
AG, Oberkochen, Germany). In order to visualise cell boundaries
within spores, spore-suspensions (106 spores ml 1 in sterile distilled water) were stained for five minutes at room temperature
with the fluorescent cell wall stain calcofluor white at a 1% concentration and imaged using the same microscope with a 405 nm
laser. Distance between septa and number of individual cells in
each spore were then assessed using ImageJ software (Schneider
et al., 2012). Spores from three replicate cultures were included
in these analyses and for each replicate culture a minimum of 11
spores were assessed. Differences in mean cell length and number
of spores between strains were assessed using ANOVA on
log2e -transformed data treating each individual spore as a technical
replicate and each culture as a biological replicate. This was followed by Fisher’s LSD test to determine significant pairwise differences between strains. To determine differences in mean
proportion of spores with only one cell present between strains,
two sample binomial tests were used to compare the WT with each
individual strain, DZtCBR1-1 and DZtCBR1-2. Statistical analyses
were carried out using the GenStat (2014, 17th edition, Ó VSN
international Ltd, Hemel Hempstead, UK) statistics package.
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2.7. GC–MS analysis of sterol content of DZtCBR1-1
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Samples from four independent liquid cultures were analysed.
Each sample of 20 mg fresh weight (freeze-dried) was used for
replicate analyses and results from WT and the DZtCBR1-1 mutant
strain were compared using Student’s t-tests. Non-saponifiable
lipids were extracted as reported previously (Kelly et al., 1995).
Samples were dried in a vacuum centrifuge (Heto) and derivatized
by addition of 100 ll of 90% bis(trimethylsilyl)-trifluoroacetamide
(BSTFA) – 10% trimethylsilyl (TMS) (Sigma–Aldrich) and 200 ll
anhydrous pyridine (Sigma–Aldrich) and heating for 2 h at 80 °C.
Gas chromatography–mass spectrometry was performed using a
VG12-250 mass spectrometer (VG Biotech) with splitless injection.
Individual sterols were identified by reference to relative retention
times, mass ions, and fragmentation patterns. Data were analysed
using MSD Enhanced ChemStation (Agilent Technologies).
Statistical analyses were carried out using the GenStat (2014,
17th edition, 297 Ó VSN international Ltd, Hemel Hempstead,
UK) statistics package.
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2.8. GC–MS analysis of fatty acid methyl ester content of DZtCBR1-1
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Samples from five independent liquid cultures were analysed.
Each sample of 20 mg fresh weight was used for replicate analyses,
and results from WT and the DZtCBR1-1 mutant strain were
compared using Student’s t-tests. Lipids were extracted and
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methylated as described (Garces and Mancha, 1993) with minor
modifications. Methyl heptadecanoate (C17:0) was added to samples as an internal standard. Following methylation the heptane
fraction was concentrated and re-suspended in 300 ll solvent prior
to injection of 1 ll onto the GC column. Methyl ester derivatives of
total fatty acids extracted were analysed by GC (Agilent 7890A)
using an Agilent DB-225 column (30 m 0.32 mm 0.3 lm).
Inlet and detector temperature was set to 250 °C and 1 ll of each
sample was analysed using splitless injection and a constant flow
rate of 2 ml min 1. The oven temperature cycle was set as follows:
a start temperature of 50 °C was held for 1 min to allow vaporised
samples and the solvent (hexane) to condensate at the front of the
column. Oven temperature was then increased rapidly to 190 °C at
a rate of 40 °C min 1 followed by a slower increase to 220 °C at a
rate of 1.5 °C min 1. The final temperature of 220 °C was held for
1 min giving a total run time of 25 min 50 s per sample. Fatty acid
methyl esters (FAMEs) were detected using a Flame Ionisation
Detector (FID). Chromatograms were analysed using the offline
session of the Agilent ChemStation software (Agilent
Technologies). The retention time and identity of each fatty acid
methyl ester (FAME) peak was calibrated using the FAME Mix
Rapeseed oil standard (Supelco). Statistical analyses were carried
out using the GenStat (2014, 17th edition, Ó VSN international
Ltd, Hemel Hempstead, UK) statistics package.
381
2.9. HPLC analysis of sphingolipid long chain 320 base content of
DZtCBR1-1
405
Samples from four independent liquid cultures were analysed.
Each sample of 20 mg fresh weight was used for replicate analyses
and results from WT and the DZtCBR1-1 mutant strain were compared using a Student’s t-test. A 2 lg aliquot of d20:0 LCB was used
as the internal standard. Long chain bases (LCBs) were liberated
from material by an alkaline hydrolysis extraction method based
on (Sperling et al., 1998). Briefly, this was performed using 10%
BaOH and dioxane 1:1 v/v in capped tubes overnight at 110 °C.
These were then cooled and extracted with chloroform/dioxane/water (8/3/8, v/v/v). The LCB fraction was converted to
dinitrophenyl derivatives with 0.2 ml 0.5% (v/v) methanolic
1-fluoro-2,4-dinitrobenzene and 0.8 ml 2 M boric acid/KOH at
60 °C for 30 min. LCBs were then extracted by phase partitioning
with CHCl3/methanol/H2O, 2:1:1 (v/v/v). The organic phase was
removed and washed with an equal volume of 0.1 M KOH and
0.5 M KCl. The organic phase was then blown down and resuspended in 200 ll MeOH for analysis. Analysis by reverse-phase
HPLC was performed using a C18 RP 250 4 mm column with a
flow rate of 1 ml min 1 and a concave gradient from 80% to 100%
methanol/acetonitrile/2-propanol, 10:3:1 (v/v/v), against water in
45 min. The elution was monitored with ESI-MS/MS MRM on a
4000 QTRAP and at a wavelength of 350 nm on an Agilent 1200
HPLC. Statistical analyses were carried out using the GenStat
(2014, 17th edition, Ó VSN international Ltd, Hemel Hempstead,
UK) statistics package.
407
3. Results
432
3.1. The Z. tritici genome encodes three putative microsomal CBRs,
alike many other genomes of filamentous fungi
433
In order to investigate the presence of putative microsomal CBR
sequences in 21 fungal genomes derived from pathogens and
non-pathogens, in both the Basidiomycete and Ascomycete phyla,
BLASTp analyses were conducted using the S. cerevisiae CBR
sequence ScCBR1 as a query. Sequences retrieved were subjected
to a TargetP analysis to determine whether the predicted proteins
435
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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were likely to be localised to mitochondria or microsomes. This
analysis identified three putative microsomal CBR proteins in Z.
tritici. The putative microsomal CBR sequences were named
ZtCBR1-3 in order of similarity to SsCBR1 (GenBank accessions:
XP_003854385.1, XP_003852872.1 and XP_003847738.1; and
Ensembl identifiers: Mycgr3T69942, Mycgr3T57682 and
Mycgr3T51378, respectively for ZtCBR1, ZtCBR2 and ZtCBR3).
These three sequences contained both NAD- and FAD-binding
domains canonical for CBRs (Fig. 1); the ZtCBR2 sequence also contained a cytochrome b5 fusion domain (PFam identifier: PF00173)
at the N-terminus (Table 1 and Fig. 1).
This higher number of microsomal CBR sequences relative to
S. cerevisiae was also seen amongst distantly related fungi. For
example the non-pathogenic fungus Aspergillus nidulans contained
seven putative microsomal CBRs, the highest number observed for
the fungal species analysed in this study. The lowest number of
putative microsomal CBRs found was in Mycosphaerella musiva, a
plant pathogenic Dothideomycete fungus related to Z. tritici which
contained only a single CBR sequence lacking a TargetP-predicted
mitochondrial localisation. All sequences identified that were
predicted to be localised to mitochondria through TargetP analyses
showed higher similarity to SsMCR1 than SsCBR1, which was
initially used to retrieve them. In the species A. terreus,
Colletotrichum
graminicola,
Dothistroma
septosporum
and
Mycosphaerella fijiensis, two to three CBR sequences with
TargetP-predicted mitochondrial localisation and higher similarity
to SsMCR1 were retrieved using SsCBR1 as a query sequence. In
addition to NAD and FAD-binding domains, PFam analyses also
identified b5 fusion domains in a number of putative microsomal
CBR sequences other than ZtCBR2; these were all present at the
N-terminus.
No obvious link between number of CBR sequences and fungal
lifestyle was apparent except for the lack of CBR sequences
observed in four of the five endophytic fungal species analysed
(Table 1). The data suggest that many filamentous fungi (perhaps
excluding certain endophytic species) have a greater diversity of
CBRs relative to the Ascomycete yeast S. cerevisiae.
3.2. RNA sequencing analysis demonstrates high constitutive
expression of ZtCBR1 and lower expression with transient upregulation of ZtCBR2 in planta
In order to identify genes that might be important for infection,
an RNA-seq analysis was conducted in a previous study (for details
see Finn et al., 2014) on samples taken from two in vitro growth
conditions and five in planta infection time points including one,
four, nine, 14 and 21 days post inoculation (DPI). These time points
are representative of major transitions in fungal growth; day one
and day four representing the symptomless phase, day nine representing the transition to necrosis, day 14 necrotrophic growth and
day 21 asexual sporulation. Analysis of mean FPKM values showed
that ZtCBR1 was highly expressed in both in vitro conditions and at
all tested infection time points (Fig. 2A). ZtCBR2, though generally
less expressed overall, was up-regulated in the nutrient-rich
ZtCBR1
ZtCBR2
ZtCBR3
FAD_binding
Cyt_b5
FAD_binding
medium PDB (p < 0.05) relative to during growth in CDB, and again
(although data were not significant) on day four of infection
relative to during growth in CDB. In addition, two genes neighbouring ZtCBR2 in the Z. tritici genome, annotated as a predicted CYP,
ZtCYP-24 (GenBank accession: XP_003853538.1), and a predicted
hydroxyacyl
coA
dehydrogenase
(GenBank
accession:
XP_003852872.1), showed similar expression profiles to ZtCBR2.
Both genes exhibited a significant up-regulation in PDB (p < 0.05)
and up-regulation on day four of infection (p < 0.05) either relative
to during growth in CDB for ZtCYP-24 or to all other infection time
points for the hydroxyacyl CoA dehydrogenase (Fig. 2B). ZtCBR3
displayed a similar (albeit lower level) expression pattern to
ZtCBR2 and was significantly up-regulated during growth in PDB
relative to during growth in CDB (p < 0.05) (Fig. 2A).
493
3.3. Fungal gene deletion and wheat leaf infection assays demonstrate
an important role for DZtCBR1 in disease progression and asexual
sporulation in Z. tritici
507
Based on the previous gene expression analysis, and in order to
assess the roles for different CBRs and CYPs in fungal growth and
virulence, various gene disruption strains were generated in the
DKu70 strain of Z. tritici IPO323 and PCR-verified (Supplementary
Fig. 1). These were then tested for the ability to cause disease on
the STB-susceptible wheat cultivar Riband (Keon et al., 2007).
Two independent CBR1 mutants, DZtCBR1-1 and DZtCBR1-2,
both caused delayed symptom manifestation in planta. At 11 DPI
when symptoms first appeared in the WT, neither DZtCBR1-1 nor
DZtCBR1-2 had caused any symptoms. At 14 DPI when the WT
had caused substantial host necrosis, only limited chlorosis was
apparent in leaves infected with DZtCBR1-1 and DZtCBR1-2. After
21 days, leaf necrosis and pycnidiation were apparent in the WT
but only patchy chlorosis was apparent in DZtCBR1-1 and
DZtCBR1-2. After 30 days, necrosis was observed in leaves inoculated with DZtCBR1-1 and DZtCBR1-2, though pycnidia were not
visible (Fig. 3A). In contrast to wild-type infections, no pycnidia
were visualised on DZtCBR1-infected leaves even when assays
were allowed to proceed for a further 14 days (at 44 DPI – data
not shown). Quantitative analysis of asexual sporulation
performed at 34 DPI, demonstrated that both DZtCBR1-1 and
produced
significantly
reduced
asexual
DZtCBR1-2
spore-numbers (p < 0.05) relative to the WT (Fig. 3B).
In contrast all other mutant strains except for DZtCBR1-1 and
DZtCBR1-2 caused WT symptoms in planta with no changes in
asexual spore counts (Supplementary Fig. S2). After 14 days cell
death was apparent in leaves inoculated with WT, DZtCYP-24 and
DZtCBR2 strains. After 21 days symptoms had progressed to
widespread necrosis at the site of inoculation, and necrotic lesions
contained numerous pycnidia. For all strains a total of 12 leaves
were evaluated, each showing symptoms consistent with the next.
Two representative leaves are shown for each assay in
Supplementary Fig. S2, A. Asexual sporulation did not appear to
be affected by functional ablation of ZtCYP-24 and ZtCBR2, as
510
313
NAD_binding
FAD_binding
NAD_binding
NAD_binding
488
309
Fig. 1. Structural characteristics of the three putative Z. tritici microsomal CBR proteins. PFam domains of the three Z. tritici putative microsomal CBR sequences retrieved.
Each predicted protein sequence contains FAD-binding (PFam identifier: PF00667) and NAD-binding (PFam identifier: PF08030) domains canonical for CBR sequences. ZtCBR2
also contains a b5-fusion domain (PFam identifier: PF00173) at the N-terminus. Amino acid sequence length is given to the right.
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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541
542
543
TargetP
mitochondrial prediction?
ScMCR1 aa
identity (%)
ScMCR1 e
value
b5 fusion
domain?
Fungal lifestyle
XP_003854385.1 (Ztcbr1)
XP_003852872.1 (Ztcbr2)
XP_003852910.1
XP_003847738.1 (Ztcbr3)
XP_756793.1
XP_759922.1
XP_001799967.1
XP_001806619.1
XP_001801691.1
XP_003322373.1
XP_003319934.2
CCA68189.1
CCA67532.1
XP_009850995.1
XP_009854993.1
EGZ77533.1
XP_009856344.1
XP_009849163.1
XP_956601.1
XP_965191.1
XP_964971.1
XP_961775.1
XP_007925194.1
XP_007926128.1
XP_007922376.1
XP_385028.1
ESU10740.1
XP_383723.1
XP_382313.1
XP_381102.1
XP_387123.1
XP_385079.1
EME45766.1
EME46111.1
EME38673.1
EFQ24978.1
EFQ25098.1
EFQ27839.1
EFQ36452.1
XP_007700426.1
XP_007700318.1
XP_007704621.1
EMD92868.1
EMD88525.1
EMD86858.1
EPQ65715.1
CCU81251.1
EPQ62420.1
CCU78450.1
XP_001208762.1
XP_001218611.1
XP_001215899.1
Q0CRD8.2
XP_001212924.1
Zymoseptoria tritici
Zymoseptoria tritici
Zymoseptoria tritici
Zymoseptoria tritici
Ustilago maydis
Ustilago maydis
Stagonospora nodorum
Stagonospora nodorum
Stagonospora nodorum
Puccinia graminis
Puccinia graminis
Piriformospora indica
Piriformospora indica
Neurospora tetrasperma
Neurospora tetrasperma
Neurospora tetrasperma
Neurospora tetrasperma
Neurospora tetrasperma
Neurospora crassa
Neurospora crassa
Neurospora crassa
Neurospora crassa
Mycosphaerella fijiensis
Mycosphaerella fijiensis
Mycosphaerella fijiensis
Fusarium graminearum
Fusarium graminearum
Fusarium graminearum
Fusarium graminearum
Fusarium graminearum
Fusarium graminearum
Fusarium graminearum
Dothistroma septosporum
Dothistroma septosporum
Dothistroma septosporum
Colletotrichum graminicola
Colletotrichum graminicola
Colletotrichum graminicola
Colletotrichum graminicola
Cochliobolus sativus
Cochliobolus sativus
Cochliobolus sativus
Cochliobolus heterostrophus
Cochliobolus heterostrophus
Cochliobolus heterostrophus
Blumeria graminis
Blumeria graminis
Blumeria graminis
Blumeria graminis
Aspergillus terreus
Aspergillus terreus
Aspergillus terreus
Aspergillus terreus
Aspergillus terreus
49
39
39
33
46
40
51
46
39
48
38
49
32
50
46
42
41
32
50
46
42
33
44
40
30
46
46
43
42
39
32
30
44
38
33
50
46
41
33
54
44
40
53
44
40
45
44
43
42
51
45
44
41
39
1.00E
1.00E
3.00E
1.00E
6.00E
8.00E
5.00E
2.00E
3.00E
5.00E
1.00E
6.00E
1.00E
1.00E
2.00E
1.00E
2.00E
2.00E
2.00E
4.00E
1.00E
6.00E
9.00E
3.00E
2.00E
1.00E
4.00E
9.00E
4.00E
2.00E
1.00E
4.00E
5.00E
1.00E
5.00E
1.00E
8.00E
2.00E
1.00E
5.00E
2.00E
8.00E
8.00E
2.00E
7.00E
3.00E
2.00E
2.00E
3.00E
7.00E
1.00E
1.00E
3.00E
5.00E
N
N
Y
N
N
Y
N
N
Y
N
Y
N
Y
N
N
Y
N
N
N
N
Y
N
N
Y
Y
N
N
N
N
Y
N
N
N
Y
Y
N
N
Y
Y
N
N
Y
N
N
Y
N
N
N
N
N
N
N
Y
Y
–
–
47
–
–
36
–
–
46
–
40
–
46
–
–
49
–
–
–
–
43
–
–
45
32
–
–
–
–
46
–
–
–
42
31
–
–
46
34
–
–
46
–
–
46
–
–
–
–
–
–
–
49
48
–
–
2.00E
–
–
3.00E
–
–
5.00E
–
6.00E
–
2.00E
–
–
3.00E
–
–
–
–
1.00E
–
–
2.00E
2.00E
–
–
–
–
2.00E
–
–
–
3.00E
4.00E
–
–
2.00E
3.00E
–
–
2.00E
–
–
2.00E
–
–
–
–
–
–
–
2.00E
7.00E
N
Y
N
N
N
N
N
Y
N
N
N
N
N
N
Y
N
N
N
N
Y
N
N
N
N
N
Y
Y
Y
Y
N
N
N
N
N
N
N
Y
N
N
N
Y
N
N
Y
N
N
N
N
N
N
Y
Y
N
N
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Biotrophic plant pathogen
Biotrophic plant pathogen
Necrotrophic plant pathogen
Necrotrophic plant pathogen
Necrotrophic plant pathogen
Biotrophic plant pathogen
Biotrophic plant pathogen
Endophyte
Endophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Saprophyte
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Hemibiotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Nectrotrophic plant pathogen
Biotrophic plant pathogen
Biotrophic plant pathogen
Biotrophic plant pathogen
Biotrophic plant pathogen
Facultative parasite/saprophyte
Facultative parasite/saprophyte
Facultative parasite/saprophyte
Facultative parasite/saprophyte
Facultative parasite/saprophyte
82
63
57
30
82
41
95
70
51
84
47
93
46
80
72
53
53
32
80
72
53
31
80
55
29
71
59
69
65
56
34
27
84
48
37
81
72
57
33
89
69
53
89
69
53
66
64
58
58
88
74
69
49
47
78
62
76
62
69
84
83
73
50
75
69
46
76
53
76
76
86
83
No. of Pages 16, Model 5G
ScCBR1 e
value
YFGBI 2846
ScCBR1 aa
identity (%)
12 June 2015
Species
M.C. Derbyshire et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx
Sequence
6
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
Table 1
Number and distribution of Saccharomyces cerevisiae CBR homologues in filamentous fungal genome sequences Below left: four plant endophytic fungi that had no sequences homologous to S. cerevisiae CBRs in their genomes.
YFGBI 2846
No. of Pages 16, Model 5G
12 June 2015
Microsomal
Mitochondrial
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
parasite/saprophyte
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
Facultative
a
Endophytes with no sequences retrieved by SsCBR1
Epichloe festucae
Ascocoryne sarcoides
Penicillium aurantiogriseum
Harpophora oryzae
Fungal lifestyle source, Urban et al., 2015 Nucleic Acids Research. Names given to the Z. tritici CBR genes referred to throughout the rest of this study are given to the right.
N
N
N
Y
Y
N
Y
Y
N
N
Y
Y
N
1.00E 67
–
–
–
–
1.00E 86
–
–
–
–
–
–
5.00E 79
39
–
–
–
–
48
–
–
–
–
–
–
47
Y
N
N
N
N
Y
N
N
N
N
N
N
Y
31
88
88
77
77
57
59
38
31
86
75
70
50
8.00E
6.00E
7.00E
3.00E
5.00E
4.00E
5.00E
6.00E
9.00E
2.00E
4.00E
1.00E
4.00E
31
51
51
47
47
43
38
37
32
49
45
44
43
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
XP_001214268.1
XP_663970.1
Q5AZB4.2
XP_661466.1
CBF75218.1
XP_658036.1
XP_682189.1
CBF82301.1
XP_663990.1
XP_755738.2
XP_753636.1
XP_748717.1
XP_750202.1
Sequence
Table 1 (continued)
Species
terreus
nidulans
nidulans
nidulans
nidulans
nidulans
nidulans
nidulans
nidulans
fumigatus
fumigatus
fumigatus
fumigatus
ScCBR1 aa
identity (%)
ScCBR1 e
value
TargetP
mitochondrial prediction?
ScMCR1 aa
identity (%)
ScMCR1 e
value
b5 fusion
domain?
Fungal lifestyle
M.C. Derbyshire et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx
7
evidenced by retrieval of a WT amount of asexual spores from
infected leaves after 21 days (Supplementary Fig. 2B).
544
3.4. DZtCBR1 spores have altered morphology and transition more
slowly to filamentous growth
546
In order to capture morphological defects observed from bright
field and laser scanning microscopic imaging of DZtCBR1 strains
(Fig. 4A and B), both individual cell length (defined as distance
between septa in the multicellular spores) and the number of cells
per spore were assessed. These microscopic analyses revealed that
both DZtCBR1 strains exhibited a significant overall decrease in
individual
cell
length
(mean
length,
WT = 11.14 lm,
DZtCBR1-1 = 7.17 lm, DZtCBR1-2 = 7.97 lm, p < 0.05) and increase
in the proportion of single-celled spores (binomial test DZtCBR1-1
vs WT, p = 0.003, DZtCBR1-2 vs WT p < 0.001) (Fig. 4C and D).
In order to infect wheat leaves, Z. tritici spores require differentiation into hyphae. It is thought that hyphal growth is induced
when Z. tritici is exposed to low nutrient environments such as
on the leaf surface, or in sterilised water culture and water agar
in vitro. In order to assess defects in hyphal growth in both
DZtCBR1 strains, spores were spot inoculated onto 1% water agar
plates and grown for two weeks. After four days WT spores had
formed an extensive hyphal network, whereas DZtCBR1 spores
had produced no hyphae. However, after six days limited hyphal
growth was observed for both DZtCBR1 strains, and after two
weeks the mycelium exhibited almost WT filamentous growth
(Fig. 5).
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3.5. DZtCBR1-1 exhibits an altered fatty acid methyl ester profile
570
In order to assess whether ZtCBR1 ablation had impacted on
fatty acid biosynthesis by fungal cells, the fatty acid methyl ester
(FAME) profile of the WT strain and DZtCBR1-1 were analysed
using GC–MS. DZtCBR1-1 displayed a significant decrease in relative abundance of the fatty acid species 16:1, 18:0 and 18:1
(p < 0.001). This strain also displayed a significant increase in relative abundance of the polyunsaturated species 18:2 (p < 0.001).
The relative abundances of the species 16:0 and 18:3 were not significantly changed relative to WT relative abundances (p > 0.05)
(Fig. 6A). The total amount of FAMEs in DZtCBR1-1 was not significantly altered relative to WT levels (p > 0.05) (Fig. 6B).
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3.6. DZtCBR1-1 displays an altered sphingolipid long chain base (LCB)
profile
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In order to assess the effect of ZtCBR1 ablation on sphingolipid
biosynthesis, sphingolipid LCBs were analysed in the WT strain
and DZtCBR1-1 using HPLC. The DZtCBR1-1 mutant exhibited a significant decrease in relative abundance of the LCB species dihydroxy 19:2 (d19:2) and dihydroxy 18:0 (d18:0) compared to the
WT (p < 0.01). This strain also displayed an increase in the relative
abundance of trihydroxy 18:0 (t18:0) that was approaching significance at the 5% level (p = 0.054) (Fig. 7).
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3.7. DZtCBR1-1 displays an altered sterol profile
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In order to assess the effect of ZtCBR1 ablation on sterol biosynthesis, the sterol profile of the WT and DZtCBR1-1 strains were
analysed using GC–MS. DZtCBR1-1 displayed a significant reduction in the relative abundance of the final product of the sterol
pathway, ergosterol (ergosta-5,7,22-trienol), relative to the WT
(p < 0.001). Several intermediate compounds in the sterol biosynthesis
pathway
including
ergosta-5,8,22,24(28)-tetraenol,
ergosta-5,8,22-trienol, ergosta-7,22-dienol and obtusifoliol (14adimethyl-5a-ergosta-8,24(28)-dienol) were significantly increased
593
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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(A) 200
ZtCBR1
180
ZtCBR2
Mean FPKM
160
ZtCBR3
*
140
120
100
80
*
60
*
40
20
0
CDB
PDB
1
4
In vitro medium
(B)
9
14
21
DPI
450
*
400
Mean FPKM
350
300
250
ZtCYP24
200
ZtCBR2
Hydroxyacyl CoA
**
150
dehydrogenase
100
* *
50
0
CDB
PDB
1
4
In vitro medium
9
14
21
DPI
(C)
ZtCYP 24
ZtCBR2
Hydroxyacyl CoA
dehydrogenase
6746 bp
Fig. 2. Expression profiles of CBR and related genes in Z. tritici. (A) Mean FPKM values showing expression of Z. tritici CBRs in Czapek Dox broth (CDB), potato dextrose broth
(PDB) and at one, four, nine, 14 and 21 days post inoculation (DPI) of wheat leaves; significant differences in expression of ZtCBR2 and ZtCBR3 (* p < 0.05) were found during
growth in PDB relative to during growth in CDB. (B) Mean FPKM values showing expression profile of ZtCBR2 and the two neighbouring genes, ZtCYP-24 and a putative
hydroxyacyl CoA dehydrogenase across the same set of conditions. (C) A diagram showing gene organisation across the region with the total size of the putative three gene
cluster in base pairs indicated below. The two genes neighbouring ZtCBR2 were significantly up-regulated both in PDB and on day four of infection (* p < 0.05) relative to
during growth in CDB; ZtCBR2 exhibited a similar expression profile though apparent up-regulation was only significant in PDB (p < 0.05).
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in relative abundance in the DZtCBR1-1 strain compared to the WT
(p < 0.01, p < 0.01, p < 0.001 and p < 0.01 respectively). The substrate of the enzyme CYP51 (eburicol (4,4,14-trimethylergos
ta-8,24(28)-dienol)), which is a target of azole antifungals, accumulated in DZtCBR1-1 but was not detected in the WT (p < 0.001)
(Fig. 8).
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4. Discussion
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4.1. Unlike S. cerevisiae many filamentous fungal genomes have more
than one microsomal CBR sequence
In several eukaryotes including plants and animals, the microsomal CBR-b5 electron transfer system has been shown to be
important for the function of desaturase and hydroxylase enzymes
involved in the biosynthesis of unsaturated fatty acids (UFAs), sterols and sphingolipids (Sperling et al., 1998; Uttaro, 2006;
Poklepovich et al., 2012). Additionally, it is thought to be involved
in certain CYP-catalysed reactions (Lamb et al., 1999, 2001). The
genome of the model yeast S. cerevisiae contains only a single copy
microsomal CBR sequence (Csukai et al., 1994; Truan et al., 1994).
In the previously analysed filamentous fungus M. alpina, which is
used in the industrial production of arachidonic acid, two microsomal CBRs have been identified in biochemical studies and structurally characterised (Sakuradani et al., 1999; Certik et al., 1999),
though their functional importance to the organism is not known
and the biochemical role of the secondary CBR is also unclear.
In the current study putative microsomal CBR sequences
encoded in a range of fully sequenced fungal genomes were identified from a number of distantly related fungi including plant
pathogens (biotrophs, hemibiotrophs and necrotrophs) and saprophytes. The mean number of putative microsomal CBR sequences
identified was three, as was observed for Z. tritici, though some
species contained considerably more. For instance A. nidulans contained seven and Fusarium graminearum contained six copies.
However others, including M. fijiensis and D. septosporum, were
more similar to S. cerevisiae having only a single predicted microsomal CBR sequence (Table 1). This is perhaps a little surprising
given that these two fungi are also Dothideomycetes and are members of the genus Mycosphaerellaceae alongside Z. tritici.
Intriguingly out of the five endophytic species analysed only the
genome of one contained sequences similar to the S. cerevisiae
CBRs. Though it is not possible to identify the precise reason for
a larger number of microsomal CBR enzymes in certain fungal
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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(B)
(A)
Spores per 5 cm inoculated leaf length
900000
11 DPI
14 DPI
800000
700000
600000
500000
400000
300000
200000
100000
0
1
*
*
2
3
Strain
21 DPI
Mock
30 DPI
Fig. 3. DZtCBR1 mutants show delayed disease symptom induction and strongly reduced asexual sporulation on wheat leaves. (A) Leaves infected with WT, DCBR1-1 and
DZtCBR1-2 after 11, 14, 21 and 30 DPI; mock-inoculated control leaves at 30 DPI are shown to the right. A total of eight leaves per strain/mock were inoculated and two
representative leaves are shown. (B) Mean number of spores (recovered by washing) per 5 cm length of inoculated leaf for WT, DZtCBR1-1 and DZtCBR1-2 after 34 DPI
showing a significant reduction in asexual sporulation of the mutant strains relative to the WT (* p < 0.05).
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species without further functional studies, we could speculate that
this larger number might be associated with a larger diversity of
the terminal CBR electron acceptors. In particular the CYPs are
known to be highly diversified amongst fungi, where they are
thought to be important for metabolism of the diverse array of
xenobiotics to which the organism may be exposed (Chen et al.,
2014). It is interesting to note that the largest number of microsomal CBRs was found in the species A. nidulans, which is capable of
colonising numerous environmental niches and therefore may
require the ability to metabolise a more diverse array of xenobiotic
compounds.
Our genome-wide analysis overall suggested no clear links
between the numbers of predicted CBR genes with either particular
pathogenic or saprophytic lifestyles. The exception to this is the
five endophytic species analysed which frequently returned no significant sequence homologues (highest e value cut off used = e 10).
Contrarily for one of these species, the rice endophyte Harpophora
oryzae, the predominant difference between its genome and those
of non-endophytic species that has been observed is relative
expansion of numerous gene families, in particular those associated with transposable elements and carbohydrate metabolism.
In fact, in this species only 10 gene families were found to be contracted relative to non-endophytes (though details of these
families are not presented in the cited study) (Xu et al., 2014).
Though only a speculation, it is possible that the selective pressures of an endophytic lifestyle may lead to a loss of genes associated with lipid metabolism like the CBRs. However, such an
observation has yet to be formally tested.
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4.2. ZtCBR1 is constitutively expressed and required for full virulence
on wheat leaves and asexual sporulation whereas ZtCBR2 is not
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In addition to the presence of canonical CBR domains in the
retrieved sequences, PFam analysis also identified numerous putative microsomal CBRs with N-terminal b5 fusions. Though such
fusions have been reported before (Yantsevich et al., 2008; Davis
et al., 2002), little is known about their functions. ZtCBR2 in Z. tritici
was found to contain a b5 fusion domain. Intriguingly this gene
shared a similar expression pattern to two neighbouring genes,
including a putative CYP, annotated in the Z. tritici genome
sequence as CYP-24, and a putative hydroxyacyl coA dehydrogenase
(Fig. 2B). Given the presence of a b5 fusion domain and the observation that the CBR-b5 system may transfer electrons to CYPs, it is
possible that this represents a discrete, co-regulated electron
transfer chain.
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Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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WT
(A)
ΔZtCBR1-1
ΔZtCBR1-2
50 μm
(B)
WT
∆ZtCBR1
10 μm
*
*
Strain
% Single-celled spores
Cell length (mm)
(C)
**
***
Strain
Fig. 4. DZtCBR1 mutants show abnormal spore morphologies. (A) Bright field imaging of WT and DZtCBR1 spores. Arrowheads highlight examples of single-celled spores
more frequently observed in DZtCBR1. (B) Representative spores of WT and DZtCBR1 strains stained with calcofluor white. (C) Mean cell length for WT and two DZtCBR1
strains. Bars represent standard error (* p < 0.05). (D) Mean percentage of single-celled spores for WT and two DZtCBR1 strains. Bars represent standard error (** p < 0.01,
***
p < 0.001).
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Genes in this putative cluster were significantly up-regulated
relative to their expression during growth in CDB in PDB and relative to either during growth in CDB or growth at all other infection
time points on the fourth day of infection (p < 0.05) (Fig. 2B and C).
Up-regulation of the genes in this cluster in the nutrient-rich medium PDB relative to CDB would suggest that this micro-region
might be involved in metabolism of complex nutrient sources.
However, low levels of expression of this cluster at later time
points during plant infection when complex nutrient sources are
released from necrotic host tissue would suggest that this is not
the case.
An alternative hypothesis would be that this cluster is responsible for degradation of a compound common to numerous plant
species, as PDB is derived from plant material. Degradation or
metabolism of host-derived molecules by CYP enzymes has been
shown to be important in various plant pathogenic fungi
(Coleman et al., 2011; Pedrini et al., 2013; Miao et al., 1991).
However, targeted deletion of the two genes ZtCBR2 and ZtCYP-24
did not lead to any reduction in virulence or asexual sporulation in
planta (Supplementary Fig. S2) or any clearly evident phenotypic
change in vitro. If ZtCBR2 is the primary electron donor for the
enzymes in this cluster, it may be that the cluster is functionally
redundant for plant infection. Furthermore, even if ZtCYP-24 is
able to receive electrons from an alternate redox partner, this
observation would indicate that alone it is not essential. Further
characterisation of this cluster, including single and double deletions of both the CYP and the putative hydroxyacyl coA dehydrogenase would be needed to characterise its potential role in plant
infection.
Despite the apparent high number and potential diversification
of CBR sequences in filamentous fungi, in Z. tritici only one CBR,
ZtCBR1, was highly expressed under both in vitro conditions and
at all infection time points tested (Fig. 2A). This may indicate that
it is the major Z. tritici CBR involved in processes described in other
eukaryotes for members of this gene family. Further evidence from
this comes from the observed biochemical and growth defects in
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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WT
Δ ZtCBR1-1
Δ ZtCBR1-2
4d
6d
14 d
10 μm
Fig. 5. DZtCBR1 mutants show reduced frequency and rate of hyphal growth. Micrograph showing the appearance of radial hyphal growth produced from the edge of a 5 ll
spore droplet for WT and the two DZtCBR1 strains after four, six and fourteen days (d) of growth on 1% water agar.
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DZtCBR1 strains and the delayed virulence and absence of asexual
sporulation in infected wheat leaves (Figs. 4–8).
4.3. ZtCBR1 is involved in fatty acid, sphingolipid and sterol
metabolism in Z. tritici
In accordance with the various roles of the CBR-b5 electron
transfer system in other eukaryotes, unsaturated fatty acid (UFA),
sphingolipid and sterol profiles of DZtCBR1 strains were analysed
and aberrations in all three of these pathways were observed
(Figs. 6–8). Biosynthesis of UFAs proceeds via the insertion of double bonds between carbons of fatty acyl chains by desaturases,
which are reliant on the CBR-b5 electron transfer system for reducing power. The first double bond is normally formed between the
9th and 10th carbons (the D9 position) of palmitic (16:0) or stearic
(18:0) acid to make palmitoleic (16:1) or oleic (18:1) acid respectively. In all eukaryotes, this is carried out by a D9 desaturase
(which is fused to b5 in fungi) that receives electrons from the
CBR system (Uttaro, 2006; Tamura et al., 1976).This is the only
desaturation event that takes place in S. cerevisiae as it only contains a single fatty acid desaturase, OLE1 (Stukey et al., 1990).
Many plants and fungi can carry out further desaturations,
including M. alpina which contains D5, D6 and D12 desaturases,
as well as a multifunctional desaturase and two additional D9
desaturases with differing substrate specificity (Knutzon et al.,
1998; Sakuradani et al., 1999; Sakuradani and Shimizu, 2003;
Kikukawa et al., 2013; MacKenzie et al., 2002; Wongwathanarat
et al., 1999). In the current study, fatty acid methyl ester (FAME)
derivatives of fatty acid species with either 16 or 18 carbons and
up to 3 double bonds were investigated in a DZtCBR1 mutant
strain, DZtCBR1-1. It was found that the relative abundances of
18:1 and 16:1 were significantly depleted in this strain, suggesting
that ZtCBR1 is an important redox partner for the Z. tritici D9
desaturase. However, the D9 desaturase substrates 18:0 and 16:0
did not significantly accumulate in DZtCBR1-1 relative to the WT.
Conversely 18:0 decreased and 18:2, the product of D12 desaturation of 18:1, increased in relative abundance in this strain (Fig. 6A).
It may be that the increase in 18:2 observed was a compensation
for the perturbations in sterol and sphingolipid biosynthesis that
were also observed in this strain. However, at this point and without protein enzyme activity studies, it is not possible to conclusively determine the precise fatty acid desaturase enzymes to
which ZtCBR1 transfers electrons.
Sphingolipid biosynthesis also involves the activity of desaturase enzymes. Sphingolipids are composed of two distinct portions, a fatty acid and a long chain base (LCB), which are
amide-linked via a variety of possible head groups. The LCB is an
aliphatic amino alcohol which may vary in the number of double
bonds or hydroxyl groups that it possesses. In addition to the
desaturases involved in LCB biosynthesis, the hydroxylases that
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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(A)
***
WT
ΔZtCBR1-1
***
***
***
16:0
16:1
18:0
18:1
18:2
18:3
FAME species
(B)
WT
∆ZtCBR1-1
Strain
Fig. 6. DZtCBR1 mutants have altered fatty acid methyl ester (FAME) profiles. (A)
Mean relative abundance of the fatty acid methyl ester (FAME) species 16:0, 16:1,
18:0, 18:1, 18:2 and 18:3 in the WT and a DZtCBR1 strain. Bars represent standard
error (** p < 0.001). (B) Total FAME content of WT and the same DZtCBR1 strain
expressed in *** lg mg 1 of fresh weight. Bars represent standard error.
WT
ΔZtCBR1-1
**
t18:0
d19:2
**
d18:0
t18:1
d18:1
LCB species
Fig. 7. DZtCBR1 mutants have altered sphingolipid profiles. (A) Mean relative
abundance of the sphingolipid long chain base (LCB) species trihydroxy 18:0
(t18:0), dihydroxy 19:2 (d19:2), dihydroxy 18:0 (d18:0), trihydroxy 18:1 (t18:1)
and dihydroxy 18:1 (d18:1) in a WT and DZtCBR1 mutant strain. Bars represent
standard error (** p < 0.01).
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are involved also rely on the CBR-b5 system for electrons. In this
study sphingolipid LCBs were analysed. Two distinct LCB desaturases are known to exist in fungi, the D4 and the D8 desaturase
(Oura and Kajiwara, 2008; Beckmann et al., 2003). It is possible
that ZtCBR1 is involved in electron transfer to these enzymes in
Z. tritici as it was found that dihydroxy 19:2 (d19:2) was depleted
in DZtCBR1-1 relative to the WT. Though there was no increase in
the precursor of this compound, d18:1, a close to statistically significant increase in the relative abundance of trihydroxy 18:0
(t18:0) was observed (p = 0.054) (Fig. 7). This may be a compensation for the lack of d19:2 as an increase in hydroxylated LCBs may
offset some of the effects of depletion of saturated LCBs. A reduction in the amount of dihydroxy 18:0 (d18:0) provides further evidence for this as it is the precursor of t18:0, which may be
produced via additional hydroxylation. However, again it is not
possible to precisely determine which sphingolipid desaturase or
hydroxylase enzymes ZtCBR1 provides electrons to without further
functional studies.
Sterol biosynthesis involves various desaturases and hydroxylases and the two cytochrome P450s (CYPs), CYP51 and CYP61
(Lepesheva and Waterman, 2007; Alcazar-Fuoli et al., 2006;
Bjorkhem and Leitersdorf, 2000; Kelly et al., 1997). S. cerevisiae
only has three CYPs, including the sterol biosynthetic CYPs and
CYP56, a dityrosine hydroxylase involved in sporulation (Briza
et al., 1994). Intriguingly CPR, the cytochrome P450 reductase, usually thought to be the primary redox partner for CYPs, has been
shown to be dispensable in S. cervisiae (Sutter and Loper, 1989).
This may be due to use of the CBR-b5 system as an alternative
redox partner for CYPs as it has been demonstrated that it can fully
support CYP51 activity in a reconstituted cell-free system (Lamb
et al., 1999). Disruption of ZtCBR1 had a major effect on sterol
biosynthesis. In DZtCBR1-1 the final product ergosterol was significantly depleted in relative abundance compared to the WT. The
two pathway intermediates that increased in relative abundance
most prominently in this strain were eburicol, the substrate of
CYP51, and obtusifoliol, the product of 4 a demethylation of eburicol by Erg25, which usually acts downstream of CYP51 (Bard et al.,
1996) (Fig. 8). This may indicate that ZtCBR1 is not only an alternative redox partner for CYP51 in Z. tritici but that it is necessary for
its function.
DZtCBR1 was also found to accumulate ergosta-5,8,22, which
has been shown to accumulate in some CYP61-inhibited fungal
strains (Loto et al., 2012). This may indicate that ZtCBR1 is also
important for the function of this enzyme in Z. tritici. Finally,
another intermediate, ergosta-7,22-dienol, was also shown to
accumulate in DZtCBR1. This is likely a result of decreased activity
of the sterol D5 desaturase enzyme, ERG3, which requires CBR for
electron transfer (Poklepovich et al., 2012; Kawata et al., 1985;
Arthington et al., 1991) (Fig. 8).
Little is understood about the direct involvement of CBR in electron transfer to the fungal sterol biosynthesis CYPs CYP51 and
CYP61, though it has been shown that disruption of the intermediate electron transfer enzyme b5 leads to an accumulation of a similar array of sterol intermediates in the Ascomycete yeast Candida
albicans (Rogers et al., 2004). Further indication of the importance
of this system for sterol biosynthesis comes from the observation
that in Schizosaccharomyces pombe, Sre1, which is known to regulate sterol biosynthetic enzymes, also regulates CBR and b5 (Todd
et al., 2006), and in humans CYP51 catalysis has been shown to
be enhanced by the presence of b5 (Lamb et al., 2001). However,
b5 has been shown to enhance CYP catalysed reactions independently of CBR via allosteric interaction (Porter, 2002), leading to
the question of potential functional redundancy between the two
electron donors CPR and CBR for CYP catalysis during sterol biosynthesis. In the current study, CBR enzyme functional ablation was
shown to strongly affect both CYP-catalysed and the D5
desaturase-catalysed steps of the sterol biosynthetic pathway,
which ultimately led to a reduction in relative abundance of the
final product ergosterol. To our knowledge this represents the first
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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(A)
WT
ΔZtCBR1-1
***
**
***
***
**
**
**
**
**
**
Sterol intermediate
(B)
4,4,14-trimethylergosta-8,24(28)-dienol (eburicol)
4,14-trimethylergosta-8,24(28)-dienol (obtusifoliol)
Erg25
CH3
CH3
H3C
CH3
CH3
CYP51
4,4-dimethylergosta-8,24(28)-dienol
H3C
CH3
Erg25
Fig. 8. DZtCBR1 mutants have altered sterol profiles. (A) Mean relative abundance of all ergosterol and all intermediates in the ergosterol biosynthetic pathway identified for
WT and a DZtCBR1 strain. Bars represent standard error, all deviations from WT levels in the DZtCBR1 strain were significant (** p < 0.01, *** p < 0.001). (B) Diagram depicting
the reactions catalysed by the enzymes CYP51 and Erg25 during sterol biosynthesis. Solid arrow represents usual direction of biosynthetic pathway. Perforated arrow
represents an alternative route for the CYP51 substrate, eburicol, which may be followed more frequently if CYP51 activity is compromised. Solid boxes surround compounds
that accumulated in DZtCBR1 relative to the WT. Red circles mark the sites of enzymatic alterations at each sterol biosynthesis step. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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direct observation of the effects of CBR functional ablation on
sterol biosynthesis in a natural live cell system.
4.4. Growth and virulence defects in DZtCBR1 strains may be
attributable to one or more of the various lipid metabolism
abnormalities
Though it may not be possible to pinpoint the precise biochemical basis of the morphological, growth and virulence defects
observed in DZtCBR1, we can speculate that alterations in sphingolipid and sterol content were a major contributory factor.
Sterols and sphingolipids are known to group together in biological
membranes to form specific regions that have been termed lipid
rafts (for a review see Simons and Sampaio, 2011). Lipid raft
regions are hypothesised to be important sites for the attachment
of specific membrane proteins involved in various cellular processes. For example, in S. cerevisiae several different sphingolipid
and sterol biosynthesis mutants have shown deficiencies in Golgi
trafficking (Proszynski et al., 2005).
Another important process in S. cerevisiae that involves
formation of lipid rafts is mating. This involves polarisation of
the plasma membrane to form a ‘schmoo tip’, in which lipid raft
domains have been observed (Bagnat and Simons, 2002). Echoing
this process, the fungus C. albicans has been shown to utilise lipid
raft domains to form hyphae. Evidence for this comes from the
observation of these regions at hyphal tips and from the formation
of aberrant hyphae in spores exposed to sterol or sphingolipid
biosynthesis-disrupting compounds (Martin and Konopka, 2004).
The morphological defects observed in DZtCBR1 strains could be
representative of an underlying defect in lipid raft formation.
Filamentous growth in this strain, though WT in appearance, was
substantially slowed (Fig. 5). Due to their reduced LCB and
ergosterol content DZtCBR1 spores may have been less frequently
able to produce lipid raft domains, leading to less frequent hyphal
extension resulting in slower overall growth. This is consistent
with observations of reductions in particular sphingolipid LCB
species and ergosterol rather than total ablation.
Other defects brought on by aberrations in lipid raft formation
may be more specific to a pathogenic lifestyle. For instance, in several mammalian-pathogenic fungi, lipid raft-embedded proteins
have been shown to be essential for adherence to host cells
(Humen et al., 2011; Mittal et al., 2008). In the plant pathogen F.
graminearum, the importance of lipid rafts in infection was demonstrated through disruption of the ceramide synthase gene (Bar1),
essential for sphingolipid biosynthesis. The F. graminearum DBar1
mutant strains were unable to produce perithecia though hyphal
differentiation and leaf penetration were still observed.
Intriguingly in the DBar1 mutant strains generated in this study,
sporulation resulted in the formation of shorter, less uniform
spores with fewer cells than the WT (Rittenour et al., 2011). A similar observation was made for the DZtCBR1 mutants presented in
the current study, which also showed these morphological defects
(Fig. 4).
The highly reduced amount of sporulation in DZtCBR1 is particularly interesting given that this strain was eventually able to produce an extensive hyphal network in vitro (Fig. 5) and induce full
necrosis of leaf tissue (Fig. 3A). This suggests that the normal virulence mechanisms that may elicit host cell death are still functionally intact in the DZtCBR1 strains but occur later, possibly as a
consequence of the reduced hyphal growth rate. There are various
possible explanations for subsequent loss of asexual sporulation in
diseased leaves, including the influence of lipid signalling on developmental processes such as growth and proliferation. For instance,
it has been demonstrated that in S. cerevisiae sphingolipid LCBs
interact with the protein kinase Pkh1, which controls numerous
processes including cell wall integrity and growth (Liu et al.,
2005). Furthermore, lipid rafts are also known to mediate localisation of H-Ras, a key element of the mitogen activated protein
kinase (MAPK) signalling pathway, to the correct sites in the cellular membrane systems (Anderson, 2006). This is intriguing because
in Z. tritici disruption of the MAPK-encoding gene MgFus3 led to a
lack of pycnidiation in vitro (Cousin et al., 2006). In light of data
presented in the current study, it is possible that this MAPK is
influenced by cellular lipid content. Future analysis should involve
sequential disruption of Z. tritici genes involved in biosynthesis of
specific lipids and lipid-derived signalling molecules.
903
4.5. Conclusion
913
By characterising members of the CBR family in Z. tritici this
study has demonstrated for the first time the importance of these
genes in regulating infection-related processes in a plant pathogenic fungus. To our knowledge, this is the first time that these
genes have been functionally characterised in fungi other than S.
cerevisiae and M. alpina. Defects in pathways thought to require
enzymes that receive electrons from CBR-b5 observed in DZtCBR1
ultimately led to an almost complete lack of asexual sporulation
in planta. Thus, processes dependent upon particular CBR-b5 electron transfers in Z. tritici may represent important new targets
for future disease intervention.
914
Acknowledgments
925
This research was carried out as part of a Biotechnology and
Biological Sciences Research Council (BBSRC) (UK) Collaborative
Award in Science and Engineering (CASE) studentship, in
collaboration with Syngenta, Jealott’s Hill, Bracknell, UK. Kim
Hammond-Kosack and Jason Rudd are supported by the BBSRC
through the Institute Strategic Program Grant 20:20 WheatÒ
(BB/J/00426X/1). All experiments involving Z. tritici WT and transgenic isolates were conducted in biological containment facilities
under FERA licence number 101948/11982851/2. This work was
supported in part by the European Regional Development
Fund/Welsh Government funded BEACON research program
(Swansea University). Many thanks to Na Li (Syngenta) for her contribution to the generation of Z. tritici mutant strains.
926
Appendix A. Supplementary material
939
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.fgb.2015.05.008.
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References
943
Alcazar-Fuoli, L. et al., 2006. Aspergillus fumigatus C-5 sterol desaturases Erg3A and
Erg3B: Role in sterol biosynthesis and antifungal drug susceptibility.
Antimicrob. Agents Chemother. 50 (2), 453–460.
Anderson, D.H., 2006. Role of lipids in the MAPK signaling pathway. Prog. Lipid Res.
45 (2), 102–119.
Arthington, B.A. et al., 1991. Cloning, disruption and sequence of the gene encoding
the yeast C-5-sterol desaturase. Gene 102 (1), 39–44.
Bagnaresi, P. et al., 2000. Tonoplast subcellular localization of maize cytochrome
b(5) reductases. Plant J. 24 (5), 645–654.
Bagnat, M., Simons, K., 2002. Cell surface polarization during yeast mating. Proc.
Natl. Acad. Sci. USA 99 (22), 14183–14188.
Bard, M. et al., 1996. Cloning and characterization of ERG25, the Saccharomyces
cerevisiae gene encoding C-4 sterol methyl oxidase. Proc. Natl. Acad. Sci. USA 93
(1), 186–190.
Beckmann, C. et al., 2003. Stereochemistry of a bifunctional dihydroceramide
Delta(4)-desaturase/hydroxylase from Candida albicans; a key enzyme of
sphingolipid metabolism. Org. Biomol. Chem. 1 (14), 2448–2454.
Bjorkhem, I., Leitersdorf, E., 2000. Sterol 27-hydroxylase deficiency: a rare cause of
xanthomas in normocholesterolemic humans. Trends Endocrinol. Metab. 11 (5),
180–183.
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
YFGBI 2846
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M.C. Derbyshire et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx
964
965
966
967
968
969
970
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
Bowler, J. et al., 2010. New capabilities for Mycosphaerella graminicola research. Mol.
Plant Pathol. 11 (5), 691–704.
Briza, P., Eckerstorfer, M., Breitenbach, M., 1994. The sporulation-specific enzymes
encoded by the DIT1 and DIT2 genes catalyze a 2-step reaction leading to a
soluble LL-dityrosine-containing precursor of the yeast spore wall. Proc. Natl.
Acad. Sci. USA 91 (10), 4524–4528.
Celik, H., Kosar, M., Arinc, E., 2013. In vitro effects of myricetin, morin, apigenin, (+)taxifolin, (+)-catechin, ( )-epicatechin, naringenin and naringin on cytochrome
b5 reduction by purified NADH-cytochrome b5 reductase. Toxicology 308, 34–
40.
Certik, M. et al., 1999. Characterization of the second form of NADH-cytochrome
b(5) reductase gene from arachidonic acid-producing fungus Mortierella alpina
1S-4. J. Biosci. Bioeng. 88 (6), 667–671.
Chen, W. et al., 2014. Fungal cytochrome P450 monooxygenases: their distribution,
structure, functions, family expansion, and evolutionary origin. Geno. Biol. Evol.
6 (7), 1620–1634.
Chomczynski, P., Sacchi, N., 1987. Single step method of RNA isolation by acid
guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162 (1),
156–159.
Coleman, J.J. et al., 2011. Characterization of the gene encoding pisatin demethylase
(FoPDA1) in fusarium oxysporum. Mol. Plant Microbe Interact. 24 (12), 1482–
1491.
Cools, H.J., Fraaije, B.A., 2008. Are azole fungicides losing ground against Septoria
wheat disease? Resistance mechanisms in Mycosphaerella graminicola. Pest
Manag. Sci. 64 (7), 681–684.
Cousin, A. et al., 2006. The MAP kinase-encoding gene MgFus3 of the nonappressorium phytopathogen Mycosphaerella graminicola is required for
penetration and in vitro pycnidia formation. Mol. Plant Pathol. 7 (4), 269–278.
Csukai, M., Murray, M., Orr, E., 1994. Isolation and complete sequence of a CBR, a
gene encoding a putative cytochrome b reductase in Saccharomyces cerevisiae.
Eur. J. Biochem. 219 (1–2), 441–448.
Davis, C.A. et al., 2002. Heterologous expression of an endogenous rat cytochrome
b(5)/cytochrome b(5) reductase fusion protein: Identification of histidines 62
and 85 as the heme axial ligands. Arch. Biochem. Biophys. 400 (1), 63–75.
do Amaral, A.M. et al., 2012. Defining the predicted protein secretome of the fungal
wheat leaf pathogen Mycosphaerella graminicola. PLoS One 7 (12), e49904.
Elahian, F. et al., 2014. Human cytochrome b5 reductase: structure, function, and
potential applications. Crit. Rev. Biotechnol. 34 (2), 134–143.
Emanuelsson, O. et al., 2000. Predicting subcellular localization of proteins based on
their N-terminal amino acid sequence. J. Mol. Biol. 300 (4), 1005–1016.
Eyal, Z., Scharen, A.L., Prescott, J.M., van Ginkel, M., 1987. The Septoria Diseases of
Wheat: Concepts and Methods of Disease Management. The International
Maize and Wheat Improvement Center, Mexico.
Finn, R.D. et al., 2014. Pfam: the protein families database. Nucleic Acids Res. 42
(D1), D222–D230.
Gan, L. et al., 2009. Role of NADPH-cytochrome P450 reductase and cytochromeb(5)/NADH-b(5) reductase in variability of CYP3A activity in human liver
microsomes. Drug Metab. Dispos. 37 (1), 90–96.
Garces, R., Mancha, M., 1993. One-step lipid extraction and fatty acid methyl esters
preparation from fresh plant tissues. Anal. Biochem. 211 (1), 139–143.
George, H.L., Hirschi, K.D., VanEtten, H.D., 1998. Biochemical properties of the
products of cytochrome P450 genes (PDA) encoding pisatin demethylase
activity in Nectria haematococca. Arch. Microbiol. 170 (3), 147–154.
Glory, M.D.D., Thiruvenangdam, 2011. Chrysin attenuates the instability of
xenobiotic metabolizing and mitochondrial enzymes during Diethyl
nitrosamine induced liver carcinoma. J. Pharm. Res. 4 (6), 1839–1842.
Gohari, A.M. et al., 2014. Molecular characterization and functional analyses of
ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria
tritici. Mol. Plant Pathol. 15 (4), 394–405.
Goodwin, S.B. et al., 2011. Finished genome of the fungal wheat pathogen
Mycosphaerella graminicola reveals dispensome structure, chromosome
plasticity, and stealth pathogenesis. PLoS Genet. 7 (6), e1002070.
Grinstead, G.F., Gaylor, J.L., 1982. Total enzymatic-synthesis of cholesterol from
4,4,14-alpha-trimethyl-5-alpha-cholesta-8,24-dien-3-beta-ol – solubilization,
resolution, and reconstitution of delta-7-sterol 5-desaturase. J. Biol. Chem.
257 (23), 3937–3944.
Hahne, K. et al., 1994. Incomplete arrest in the outer-membrane sorts NADHcytochrome-b(5) reductase to 2 different submitochondrial compartments. Cell
79 (5), 829–839.
Henderson, C.J., McLaughlin, L.A., Wolf, C.R., 2013. Evidence that cytochrome b(5)
and cytochrome b(5) reductase can act as sole electron donors to the hepatic
cytochrome p450 systems. Mol. Pharmacol. 83 (6), 1209–1217.
Huang, Y.S. et al., 1999. Cloning of Delta 12-and Delta 6-desaturases from
Mortierella alpina and recombinant production of gamma-linolenic acid in
Saccharomyces cerevisiae. Lipids 34 (7), 649–659.
Humen, M.A., Perez, P.F., Lievin-Le, V., 2011. Moal, lipid raft-dependent adhesion of
Giardia intestinalis trophozoites to a cultured human enterocyte-like Caco-2/
TC7 cell monolayer leads to cytoskeleton-dependent functional injuries. Cell.
Microbiol. 13 (11), 1683–1702.
Kawata, S., Trzaskos, J.M., Gaylor, J.L., 1985. Microsomal enzymes of cholesterol
biosynthesis from lanosterol – purification and characterization of delta-7sterol 5-desaturase of rat liver microsomes. J. Biol. Chem. 260 (11), 6609–6617.
Kelly, S.L. et al., 1995. Mode of action and resistance to azole antifungals associated
with the formation of 14-alpha-methylergosta-8,24(28)-dien-3-beta,6-alphadiol. Biochem. Biophys. Res. Commun. 207 (3), 910–915.
15
Kelly, S.L. et al., 1997. Characterization of Saccharomyces cerevisiae CYP61, sterol
Delta(22)-desaturase, and inhibition by azole antifungal agents. J. Biol. Chem.
272 (15), 9986–9988.
Keon, J. et al., 2007. Transcriptional adaptation of Mycosphaerella graminicola to
programmed cell death (PCD) of its susceptible wheat host. Mol. Plant Microbe
Interact. 20 (2), 178–193.
Kikukawa, H. et al., 2013. Characterization of a trifunctional fatty acid desaturase
from oleaginous filamentous fungus Mortierella alpina 1S-4 using a yeast
expression system. J. Biosci. Bioeng. 116 (6), 672–676.
Knutzon, D.S. et al., 1998. Identification of Delta 5-desaturase from Mortierella
alpina by heterologous expression in bakers’ yeast and canola. J. Biol. Chem. 273
(45), 29360–29366.
Kumar, R. et al., 2006. A mutation in arabidopsis cytochrome b5 reductase identified
by high-throughput screening differentially affects hydroxylation and
desaturation. Plant J. 48 (6), 920–932.
Lamb, D.C. et al., 1999. Biodiversity of the P450 catalytic cycle: yeast cytochrome
b(5)/NADH cytochrome b(5) reductase complex efficiently drives the entire
sterol 14-demethylation (CYP51) reaction. FEBS Lett. 462 (3), 283–288.
Lamb, D.C. et al., 2001. Human sterol 14 alpha-demethylase activity is enhanced by
the membrane-bound state of cytochrome b(5). Arch. Biochem. Biophys. 395
(1), 78–84.
Lee, W.S. et al., 2014. Mycosphaerella graminicola LysM effector-mediated stealth
pathogenesis subverts recognition through both CERK1 and CEBiP homologues
in wheat. Mol. Plant Microbe Interact. 27 (3), 236–243.
Lepesheva, G.I., Waterman, M.R., 2007. Sterol 14 alpha-demethylase cytochrome
P450 (CYP51), a P450 in all biological kingdoms. Biochim. Biophys. Acta-Gen.
Sub. 1770 (3), 467–477.
Liu, K. et al., 2005. The sphingoid long chain base phytosphingosine activates AGCtype protein kinases in Saccharomyces cerevisiae including Ypk1, Ypk2, and
Sch9. J. Biol. Chem. 280 (24), 22679–22687.
Loto, I., et al., 2012. Enhancement of Carotenoid Production by Disrupting the C22Sterol Desaturase Gene (CYP61) in Xanthophyllomyces dendrorhous. BMC
Microbiology. p. 12.
MacKenzie, D.A. et al., 2002. A third fatty acid Delta 9-desaturase from Mortierella
alpina with a different substrate specificity to ole1p and ole2p. Microbiol. Sgm
148, 1725–1735.
Marshall, R. et al., 2011. Analysis of two in planta expressed LysM Effector homologs
from the fungus Mycosphaerella graminicola reveals novel functional properties
and varying contributions to virulence on wheat. Plant Physiol. 156 (2), 756–
769.
Martin, S.W., Konopka, J.B., 2004. Lipid raft polarization contributes to hyphal
growth in Candida albicans. Eukaryot. Cell 3 (3), 675–684.
Miao, V.P.W., Matthews, D.E., Vanetten, H.D., 1991. Identification and chromosomal
locations of a family of cytochrome P450 genes for pisatin detoxification in the
fungus Nectria haematococca. Mol. Gen. Genet. 226 (1–2), 214–223.
Michaelson, L.V. et al., 2013. Identification of a cytochrome b5-fusion desaturase
responsible for the synthesis of triunsaturated sphingolipid long chain bases in
the marine diatom Thalassiosira pseudonana. Phytochemistry 90, 50–55.
Mirzaei, S.A., Yazdi, M.T., Sepehrizadeh, Z., 2010. Secretory expression and
purification of a soluble NADH cytochrome b5 reductase enzyme from Mucor
racemosus in Pichia pastoris based on codon usage adaptation. Biotechnol. Lett.
32 (11), 1705–1711.
Mittal, K., Welter, B.H., Temesvari, L.A., 2008. Entamoeba histolytica: lipid rafts are
involved in adhesion of trophozoites to host extracellular matrix components.
Exp. Parasitol. 120 (2), 127–134.
Moreno-Perez, A.J. et al., 2011. Sphingolipid base modifying enzymes in sunflower
(Helianthus annuus): Cloning and characterization of a C4-hydroxylase gene and
a new paralogous Delta 8-desaturase gene. J. Plant Physiol. 168 (8), 831–839.
Motteram, J. et al., 2009. Molecular characterization and functional analysis of
MgNLP, the sole NPP1 domain-containing protein, from the fungal wheat leaf
pathogen Mycosphaerella graminicola. Mol. Plant Microbe Interact. 22 (7), 790–
799.
Motteram, J. et al., 2011. Aberrant protein N-glycosylation impacts upon infectionrelated growth transitions of the haploid plant-pathogenic fungus
Mycosphaerella graminicola. Mol. Microbiol. 81 (2), 415–433.
Mutch, D.M. et al., 2007. The disruption of hepatic cytochrome P450 reductase
afters mouse lipid metabolism. J. Proteome Res. 6 (10), 3976–3984.
Neve, E.P.A. et al., 2012. Amidoxime reductase system containing cytochrome b(5)
type B (CYB5B) and MOSC2 Is of importance for lipid synthesis in adipocyte
mitochondria. J. Biol. Chem. 287 (9), 6307–6317.
Nikiforova, A.B., Saris, N.-E.L., Kruglov, A.G., 2014. External mitochondrial NADHdependent reductase of redox cyclers: VDAC1 or Cyb5R3? Free Radical Biol.
Med. 74, 74–84.
O’Driscoll, A. et al., 2014. The wheat-Septoria conflict: a new front opening up?
Trends Plant Sci. 19 (9), 602–610.
Orton, E.S., Deller, S., Brown, J.K.M., 2011. Mycosphaerella graminicola: from
genomics to disease control. Mol. Plant Pathol. 12 (5), 413–424.
Oura, T., Kajiwara, S., 2008. Disruption of the sphingolipid Delta(8)-desaturase gene
causes a delay in morphological changes in Candida albicans. Microbiol. -Sgm
154, 3795–3803.
Pedrini, N. et al., 2013. Targeting of insect epicuticular lipids by the
entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within
the context of a host-pathogen interaction. Front. Microbiol., 4
Poklepovich, T.J. et al., 2012. The cytochrome b(5) dependent C-5(6) sterol
desaturase DES5A from the endoplasmic reticulum of Tetrahymena
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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thermophila complements ergosterol biosynthesis mutants in Saccharomyces
cerevisiae. Steroids 77 (13), 1313–1320.
Porter, T.D., 2002. The roles of cytochrome b(5) in cytochrome P450 reactions. J.
Biochem. Mol. Toxicol. 16 (6), 311–316.
Proszynski, T.J. et al., 2005. A genome-wide visual screen reveals a role for
sphingolipids and ergosterol in cell surface delivery in yeast. Proc. Natl. Acad.
Sci. USA 102 (50), 17981–17986.
Richter, M.E.A. et al., 2008. Sequential asymmetric polyketide heterocyclization
catalyzed by a single cytochrome P450 monooxygenase (AurH). Angew. Chem.
Int. Ed. 47 (46), 8872–8875.
Rittenour, W.R. et al., 2011. Control of glucosylceramide production and
morphogenesis by the bar1 ceramide synthase in Fusarium graminearum. PLoS
One 6 (4), e19385.
Rogers, K.M. et al., 2004. Disruption of the Candida albicans CYB5 gene results in
increased azole sensitivity. Antimicrob. Agents Chemother. 48 (9), 3425–3435.
Rudd, J.J. et al., 2015. Transcriptome and metabolite profiling of the infection cycle
of Zymoseptoria tritici on wheat reveals a biphasic interaction with plant
immunity involving differential pathogen chromosomal contributions and a
variation on the hemibiotrophic lifestyle definition. Plant Physiol. 167 (3),
1158–1185.
Sakuradani, E., Shimizu, S., 2003. Gene cloning and functional analysis of a second
Delta 6-fatty acid desaturase from an arachidonic acid-producing Mortierella
fungus. Biosci. Biotechnol. Biochem. 67 (4), 704–711.
Sakuradani, E., Kobayashi, M., Shimizu, S., 1999. Identification of an NADHcytochrome b(5) reductase gene from an arachidonic acid-producing fungus,
Mortierella alpina 1S-4, by sequencing of the encoding cDNA and heterologous
expression in a fungus, Aspergillus oryzae. Appl. Environ. Microbiol. 65 (9),
3873–3879.
Sakuradani, E. et al., 1999. Identification of Delta 12-fatty acid desaturase from
arachidonic acid-producing Mortierella fungus by heterologous expression in
the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Eur. J.
Biochem. 261 (3), 812–820.
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of
image analysis. Nat. Methods 9 (7), 671–675.
Shockey, J.M. et al., 2005. Cloning, functional analysis, and subcellular localization
of two isoforms of NADH: cytochrome b(5) reductase from developing seeds of
tung (Vernicia fordii). Plant Sci. 169 (2), 375–385.
Siah, A. et al., 2014. QoI Resistance and Mitochondrial Genetic Structure of
Zymoseptoria tritici in Morocco. Plant Dis. 98 (8), 1138–1144.
Simons, K., Sampaio, J.L., 2011. Membrane organization and lipid rafts. Cold Spring
Harbor Perspect. Biol. 3 (10), 17.
Sperling, P., Zahringer, U., Heinz, E., 1998. A sphingolipid desaturase from higher
plants – identification of a new cytochrome b(5) fusion protein. J. Biol. Chem.
273 (44), 28590–28596.
Stukey, J.E., McDonough, V.M., Martin, C.E., 1990. The OLE1 gene of Saccharomyces
cerevisiae encodes the delta-9 fatty acid desaturase and can be functionally
replaced by the rat steroyl coA desaturase gene. J. Biol. Chem. 265 (33), 20144–
20149.
Suffert, F., Sache, I., Lannou, C., 2013. Assessment of quantitative traits of
aggressiveness in Mycosphaerella graminicola on adult wheat plants. Plant.
Pathol. 62 (6), 1330–1341.
Sutter, T.R., Loper, J.C., 1989. Disruption of the Saccharomyces cerevisiae gene for
NADPH-cytochrome-P450 reductase causes increased sensitivity to
ketoconazole. Biochem. Biophys. Res. Commun. 160 (3), 1257–1266.
Syed, K. et al., 2011. Cytochrome b(5) reductase-cytochrome b(5) as an active P450
redox enzyme system in Phanerochaete chrysosporium: a typical properties and
in vivo evidence of electron transfer capability to CYP63A2. Arch. Biochem.
Biophys. 509 (1), 26–32.
Taher, K. et al., 2014. Sensitivity of Zymoseptoria tritici isolates from tunisia to
pyraclostrobin, fluxapyroxad, epoxiconazole, metconazole, prochloraz and
tebuconazole. J. Phytopathol. 162 (7–8), 442–448.
Tamura, Y. et al., 1976. Fatty acid desaturase system of yeast microsomes –
involvement of cytochrome b5-containing electron transport chain. Arch.
Biochem. Biophys. 175 (1), 284–294.
Todd, B.L. et al., 2006. Sterol regulatory element binding protein is a principal
regulator of anaerobic gene expression in fission yeast. Mol. Cell. Biol. 26 (7),
2817–2831.
Trapnell, C. et al., 2012. Differential gene and transcript expression analysis of RNAseq experiments with TopHat and Cufflinks. Nat. Protoc. 7 (3), 562–578.
Truan, G. et al., 1994. Cloning and characterization of a yeast cytochrome b(5)encoding gene which suppresses ketoconazole hypersensitivity in a NADPHP450 reductase-deficient strain. Gene 142 (1), 123–127.
Truong, H.N., Meyer, C., Danielvedele, F., 1991. Characteristics of Nicotiana tobacum
reductase protein produced in Saccharomyces cerevisiae. Biochem. J. 278, 393–
397.
Uttaro, A.D., 2006. Biosynthesis of polyunsaturated fatty acids in lower eukaryotes.
IUBMB Life 58 (10), 563–571.
Wayne, L.L. et al., 2013. Cytochrome b5 reductase encoded by CBR1 is essential for a
functional male gametophyte in arabidopsis. Plant Cell 25 (8), 3052–3066.
Wongwathanarat, P. et al., 1999. Two fatty acid Delta 9-desaturase genes, ole1 and
ole2, from Mortierella alpina complement the yeast ole1 mutation. Microbiol. Sgm 145, 2939–2946.
Xu, X.-H. et al., 2014. The rice endophyte Harpophora oryzae genome reveals
evolution from a pathogen to a mutualistic endophyte. Sci. Rep., 4
Yantsevich, A.V., Gilep, A.A., Usanov, S.A., 2008. Mechanism of electron transfer in
fusion protein cytochrome b(5)-NADH-cytochrome b(5) reductase. Biochem.
Moscow 73 (10), 1096–1107.
Zwiers, L.H., De Waard, M.A., 2001. Efficient agrobacterium tumefaciens-mediated
gene disruption in the phytopathogen Mycosphaerella graminicola. Curr. Genet.
39 (5–6), 388–393.
Please cite this article in press as: Derbyshire, M.C., et al. Analysis of cytochrome b5 reductase-mediated metabolism in the phytopathogenic fungus
Zymoseptoria tritici reveals novel functionalities implicated in virulence. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.05.008
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