Proteomics 2006, 6, 4599–4609
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DOI 10.1002/pmic.200600052
RESEARCH ARTICLE
Identification of differentially regulated proteins in
response to a compatible interaction between the
pathogen Fusarium graminearum and its host,
Triticum aestivum
Wenchun Zhou, François Eudes and André Laroche
Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada
Using proteomic analyses, a study was carried out aimed at understanding the molecular
mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted
from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected
to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/
MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling
pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were
up-regulated, while those related to photosynthesis were less abundant following F. graminearum
infection. The DNA-damage inducible protein was found to be induced and glycosylated in
F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle
mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible
functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.
Received: January 23, 2006
Revised: April 21, 2006
Accepted: May 11, 2006
Keywords:
Fusarium graminearum / Fusarium head blight / Scab / Wheat
1
Introduction
Fusarium head blight (FHB) of wheat (Triticum aestivum L.),
caused mainly by Fusarium graminearum Schwabe [telomorph: Gibberella zeae Schw.(Petch)], is recognized as one of
Correspondence: Dr. André Laroche, Agriculture and Agri-Food
Canada, 5403 1st Avenue South, PO Box 3000, Lethbridge,
Alberta, Canada T1J 4B1
E-mail: larochea@agr.gc.ca
Fax: 11-403-382-3156
Abbreviations: CD, conserved domain; FBP, fructose-1, 6-bisphosphate; FHB, Fusarium head blight; GAPDH, glyceraldehyde-3phosphate dehydrogenase; JA, jasmonic acid; PR-protein, pathogenesis-related protein; SA, salicylic acid; SOD, superoxide dismutase
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the most destructive diseases of wheat [1, 2]. FHB results in
serious grain yield and quality losses when warm and humid
weather coincides with plant anthesis [3]. Mycotoxin contamination in harvested grain is also a major health concern
for both humans and animals [2, 4].
Wheat spikes are susceptible to F. graminearum infection
for a relatively short period of time varying from 10 to 20 days
from the beginning of anthesis through the soft dough stage
of kernel development [1]. Initial infection occurs when
ascospores (sexual spores) or macroconidia (asexual spores)
released from soil-borne debris and infected hosts are laid on
or inside flowering spikelets under appropriate or favorable
temperature and humidity conditions. A strain of F. graminearum constitutively expressing the green fluorescent protein
from jellyfish was used to study the early events of the pathogen colonization in wheat [5]. Based on those observations,
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W. Zhou et al.
fungal hyphae were visible inside the floret at the point of
inoculation within a few hours of the inoculation. The fungus
was able to spread along the spike both internally, through the
rachis, and across the external surfaces of the rachis and florets for both FHB resistant and susceptible lines [5].
Genomic approaches have been applied to characterize
wheat responses to F. graminearum infection. Identification
and molecular characterization of cDNA clones and ESTs
from F. graminearum-infected spikes revealed that transcript
levels of many pathogenesis-related (PR-) genes increased following F. graminearum infection [6–8]. Different classes of PRproteins including PR-1, PR-2 (b-1, 3 glucanases), PR-3 (chitinases), PR-5 (thaumatin-like protein), and PR-9 (peroxidases)
were induced within 6–12 h of inoculation. The proteomic
approach is a powerful tool to study plant stress response. A
global protein expression profile can be generated and compared using a 2-DE-based protein separation method and individual proteins identified when coupled to protein identification by MS technology. An initial proteomic study on the
interaction between F. graminearum and wheat reported that
proteins with antioxidant function such as superoxide dismutase (SOD), dehydroascorbate reductase, and GSTs were
up-regulated or induced 5 days after inoculation with F. graminearum, indicating an oxidative burst of H2O2 inside the
tissues infected by F. graminearum [9]. In this current study, a
systemic comparison of protein profiles among wheat spikelets inoculated with F. graminearum was made 1, 2 and 3 days
post inoculation. The objective of the current experiment was
to identify differentially accumulated proteins from both
F. graminearum and wheat involved in a compatible interaction between F. graminearum and wheat.
2
Materials and methods
2.1 Chemicals
CHAPS, IPG strips, urea, acrylamide and colloidal
CBB R250 were from Bio-Rad Laboratories Ltd. (Mississauga, ON, Canada); Sypro Ruby, Pro-Q Emerald and
ampholytes from Invitrogen Canada Inc. (Burlington, ON,
Canada); thiourea from Sigma (St. Louis, MO, USA); and
mini Complete Protease Inhibitor Cocktail Tablets from
Roche Diagnostics Canada (Laval, QC, Canada).
2.2 Preparation of conidia inoculum
The F. graminearum strain N2 used in this study was
graciously provided by Dr. Jeannie Gilbert (Cereal Research
Center, Winnipeg, Manitoba, Canada). The medium used
for conidiospore production contained 1.5% carboxymethylcellulose, 0.1% NH4NO3, 0.1% KH2PO4 monobasic,
0.05% MgSO4 ?7H2O, and 0.1% yeast extract. These constituents were thoroughly mixed while boiling, and autoclaved.
Media was inoculated with the F. graminearum isolate and
incubated on a rotary shaker (150 rpm) at 227C for 4–5 days.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 4599–4609
Prior to inoculation of plants, inoculum was filtered, rinsed
with deionized water and diluted to the desired concentration in deionized water.
2.3 Plant growth and spike inoculation
Crystal, a Canadian FHB susceptible cultivar, was used in
this study. Seeds of Crystal were planted directly in pots.
About 30 pots with four plants per pot were placed randomly
on a bench in a greenhouse maintained at 247C with a 16-h
photoperiod (artificial lights were used to maintain light
intensity over 300 mmol?m22s21 when necessary). Plants
were periodically fertilized with a solution of 20:20:20 nitrogen:phosphorus:potassium to maintain a healthy green
appearance among plants, and watered as required.
Wheat spikelets were inoculated with a suspension of
conidiospores of F. graminearum at mid-anthesis. Approximately 1000 conidiospores in a volume of 10 mL were injected into two flowering florets of a spikelet. The same volume
of deionized water was similarly injected into flowering spikelets on a different plant to serve as a control. The inoculated spikelets were marked and the time and date of inoculation recorded. Inoculated plants were placed in a mist
room immediately after inoculation where the humidity was
maintained at 95% by a computer controlled high-pressure
mist system. Temperature was increased to 267C and light
conditions were same as described above. Following inoculation, spikes were harvested 1, 2 and 3 days after inoculation. Harvested spikes were immediately placed on ice and
then, transferred into a –807C freezer for storage until protein extraction. Two complete independent biological sample
sets were analyzed in this study.
2.4 Protein extraction and quantitation
Protein samples were extracted in a cold room at 47C using
the acetone/TCA method described by Wang et al. [10] with
some modifications. Wheat spikelets inoculated with either
F. graminearum or deionized water were removed from frozen spikes with a pair of forceps. For each independent
sample set, about 15 treated or control frozen spikelets from
5 to 8 different spikes within each inoculated period were
combined into one sample and ground in a pre-chilled mortar in liquid nitrogen. Finely ground powder was collected
into a 2-mL microcentrifuge tube and weighed. One milliliter of 10% TCA, 0.07% 2-mercaptoethanol in cold (2207C)
acetone was added to 0.3 g ground tissue. The samples were
incubated for 2 h at 2207C to precipitate proteins and then
centrifuged for 20 min at 16 0006g. The pellet of precipitated proteins and debris was washed with 1 mL cold
90% acetone containing 0.07% 2 mercaptoethanol several
times until the pellet was colorless. A 10-min centrifugation
at 16 0006g was used to pellet the proteins after each wash.
Pellets were dried in a freeze vacuum dryer for 10 min, and
protein pellets were resuspended in 1 mL lysis buffer for 1 h.
The lysis buffer contained 7 M urea, 2 M thiourea,
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Proteomics 2006, 6, 4599–4609
4% CHAPS, 60 mM DTT, and 0.5% carrier ampholytes
pH 3–10; and one mini Complete Protease Inhibitor Cocktail
Tablet was added fresh into every 10 mL lysis buffer. After
centrifugation at 16 0006g for 30 min to remove debris, the
supernatant was collected, and a 5-mL sample was removed
for protein assay. The remaining supernatant was separated
into aliquots and stored at 2807C until protein electrophoresis. Protein concentration of samples was determined with
BSA for calibration of the assay [11].
2.5 IEF and SDS-PAGE
A solubilized protein sample (150 mg: analytical gels; 300 mg:
preparative gels) was mixed with lysis buffer to a total volume of 350 mL and loaded on a 17-cm pH 4–7 Bio-Rad Ready
Gel Strip with the in-gel rehydration method according to the
manufacturer’s instructions. For the second-dimension
separation, the strips were positioned on top of a 15% polyacrylamide gel in presence of SDS and sealed with 1% agarose. The gels were run for 30 min at 30 mA followed by
60 mA for 5 h using a Protean II Cell from Bio-Rad.
2.6 Staining of PAGE gels
Three staining methods were employed. The Sypro Ruby stain
method was used for staining analytical gels to obtain a linear
quantitative measurement of proteins following the instruction manual from Invitrogen Canada Inc. Images from all
Sypro Ruby-stained gels were captured using a Typhoon 9400
scanner with the same scanning settings (scan resolution:
photomultiplier: 600 V; normal sensitivity; filters: 610 BP30/
Green; 532 nm) (GE Healthcare, Baie D’Urfe, QC, Canada).
Triplicate images from three independent gels for each of the
two independent treatments were obtained for further quantitative image analyses. The colloidal CBB R250 staining method
was used for preparative gels. Protein spots with significantly
altered expression or newly induced following F. graminearum
infection were manually excised for LC-MS/MS analyses. Glycosylated proteins were stained with the Pro-Q Emerald 488
glycoprotein gel stain kit following instruction provided by
Invitrogen [12]. The same gel stained for glycoproteins were
post stained with Sypro Ruby for identification and analysis of
the whole protein pattern.
2.7 Image analysis
A computer software, Phoretix 2D Expression v2005, from
Nonlinear Dynamics (Durham, NC 27703 USA) was used to
analyze images of Sypro Ruby-stained gels. Three images for
each of the three inoculated periods, 1, 2 and 3 days after
F. graminearum infection or control (H2O) were grouped to
calculate the averaged volume of all the individual protein
spots. To reduce the experimental errors arising during process of 2-DE, a normalized volume for each individual protein spot was calculated using 100 times the volume of this
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4601
protein divided by the total volume of all proteins detected on
the same image. Warping, matching, and comparison of
volumes of proteins among the treatments were generated
by the software. Three types of proteins with altered expression due to F. graminearum infection were defined in this
experiment. Down-regulated and up-regulated proteins
means that averaged expression volumes of these proteins
over triplicate images in the F. graminearum inoculated spikelets were at least twofold lower or higher than those from
control inoculated spikelets at one or more time points. Each
independent set of sample was analyzed independently and
the spots showing consistent differentially expression pattern between F. graminearum-inoculated spikelets and control in two sets of samples were selected for further LC-MS/
MS analysis. The expression pattern and maximum fold
change for up-regulated and down-regulated spots were
averaged from two data sets. We define induced proteins for
those proteins that were only present in F. graminearuminoculated spikelets, although this kind of change theoretically belongs to a maximum up-regulation.
2.8 LC-MS/MS
Excised CBB-stained protein spots were dried by a freezer
vacuum dryer. Identification of protein was conducted by the
Stanford University Mass Spectrometry Facility (URL:
www.mass-spec.stanford.edu), using m-ESI-LC-MS/MS. The
proteins were destained and reduced with DTT, alkylated
with acrylamide and digested with trypsin (Promega, Madison, WI, USA). The resulting peptide solution was analyzed
on a Micromass CapLC and Q-TOF API US (Manchester,
UK) LC-MS system. A peptide CapTrap (Michrom Bioresources, Auburn, CA, USA) was used for online desalting,
followed by back flushing onto a 0.0756100 mm PepMap
C18 column (LC Packings, Amsterdam, The Netherlands).
Peptides were eluted from the column with a 30-min linear
gradient of 3–45% solvent B (solvent A: 97.9% H2O,
2% ACN, 0.1% formic acid; solvent B: 97.9% ACN, 2% H2O,
0.1% formic acid) at a flow rate of ,300 nL/min. The standard micromass nanospray source with blunt-tip 90-mm od,
20-mm id fused silica emitter was held at 807 C, capillary
voltage 13.4 kV, cone voltage 32 V. Data acquisition was performed in data-dependent mode, with up to three precursors
for MS/MS selected from each MS survey scan. The .DTA
files generated by Micromass ProteinLynx software were
searched against the NCBI NR protein database using the
MASCOT MS/MS Ion Search (www.matrixscience.com).
2.9 Identification of conserved domains
To determine the possible functions and classification of five
hypothetical proteins from F. graminearum, we used the
obtained sequence information to search for conserved
domains (CDs) using an on-line CD-Search software developed by Marchler-Bauer et al. [13] (www.ncbi.nlm.nih.gov/
Structure/cdd/wrpsb.cgi). The newly developed CD datawww.proteomics-journal.com
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W. Zhou et al.
base, CDD, was searched against for all possible CDs [14].
The CD with the highest score was listed as the CD for the
respective hypothetical protein.
2.10
Function characterization and subcellular
localization of proteins
The Gene Ontology Tool (www.geneontology.org) and TargetP (www.cbs.dtu.dk/services/TargetP) were used to determine functional classification and subcellular localization
prediction [15, 16]. The identification of potential glycosylation sites was carried out accessing NetOGlyc 3.1 and NetNGlyc 1.0
Servers
(www.cbs.dtu.dk/services/NetOGlyc/;
www.cbs.dtu.dk/services/NetNGlyc/) [17].
3
Results
3.1 Wheat proteins in response to F. graminearum
infection
Figure 1 shows four reproducible gel maps displaying proteins from Crystal wheat spikes 1 and 2 days post inoculation
with F. graminearum and control (H2O). Figure 2 shows the
enlarged gel images displaying the proteins from Crystal
spikes 3 days post inoculation with F. graminearum or H2O.
Numerous differentially regulated proteins were observed in
these gels. Approximately 1380 protein spots were resolved in
the pH 4–7 range on all these images. Figure 3 shows
enlarged inlets containing three different classes of proteins
that were differentially expressed due to F. graminearum
infection. A22 represents an induced protein that was detected
only in F. graminearum inoculated samples after 1 day and
beyond. A8 is an up-regulated protein. B2 represents a significantly down-regulated protein. In total, 41 proteins were
identified as being significantly altered in their expression due
to F. graminearum infection. The expression volumes of these
proteins are shown in the first column of Table 1. The maximum fold-change value of the expression volume for each
protein over the three treatment periods is listed in column 3
of Table 1. The range of fold change in the expression of all
proteins varies from 2 (sample A21) to 6.5 (sample A20). Of
the 41 protein selected, 9 down-regulated proteins were
labeled as B1–B9 in Fig. 2b. The expression volumes of these
polypeptides were significantly reduced in F. graminearuminoculated spikelets. Thirty-two induced or up-regulated proteins were labeled as A1–A24 and C1–C8 in Fig. 2a.
3.2 Identification of F. graminearum responsive
proteins
All the 41 differentially regulated proteins were analyzed by
LC-MS/MS and the best homolog for each protein is listed in
Table 1. Thirty-three polypeptides were identified as plant
proteins. Among these, protein B2 and B3 were recognized
both as a putative 40S ribosomal protein, and proteins B7,
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 4599–4609
B8 and B9 were identified as three isoforms of profilin 3.
Polypeptide A12 was annotated in the NCBI as a rice
sequence of unknown function OSJNBb0034G17.8. From a
BLAST search, its highest homolog (82% identity) was an
acetyl glutamate kinase-like protein (GenBank accession no.
CAC39078) from rice. Eight polypeptides (C1–C8) were
identified as fungal proteins and the best homolog of seven
of them were recognized as different proteins originating
from F. graminearum. Polypeptide C2 had the best homology
against a protein of unknown function from Ustilago maydis.
3.3 Functional classification and subcellular
localization prediction of F. graminearumresponsive proteins
All protein sequences detected and identified were searched
against the Gene Ontology Tool and TargetP for functional
classification and subcellular localization prediction [15, 16],
respectively. These identified proteins were found to be
involved in diverse biological processes, including defense and
stress response (A3, A4, A7, A9, A11, A17, A19, A20 and A24),
signal transduction (A2, A6, and A15), photosynthesis (A16,
A23, and B6), electron transport (A22), glycolysis (A1, B5),
protein synthesis ( B2, and B3), translation (A18), transcription
(A21), and metabolism (A5, A8, A10, A12, A13, A14, B1, B4, B7,
B8, B9). Among the up-regulated or induced proteins responsible for metabolism, two of them were related to amino acid
synthesis: A8 for tryptophan synthesis, A10 for cysteine synthesis. Glyoxalase (A5) has been reported to be involved in
detoxification of methylglyoxal, which is a cytotoxic and mutagenic compound [18]. Previous transgenic studies in tobacco
confirmed that glyoxalase is also involved in salt tolerance [19].
Alcohol dehydrogenase (A14) and glutamate dehydrogenase (A13) have important functions in carbohydrate [20] and
nitrogen metabolism [21], respectively. Two down-regulated
proteins involved in cellular metabolism are a vacuolar invertase (B1) and profilin (B7, B8, and B9). The physiological role of
vacuolar invertases appears to be diverse and recent studies
suggest that their function varies depending upon the organ/
tissue or cells in which they are expressed. Vacuolar invertases
take part in sucrose partitioning between source and sink
organs, and would be responsible for a feedback regulation of
photosynthesis [22]. Profilin is a small protein that binds to
monomeric actin (G-actin) in a 1:1 ratio, thus preventing the
polymerization of actin into filaments (F-actin). It can also
under certain circumstance promote actin polymerization [23].
These results indicate that biochemical pathways or proteins
mentioned above are affected following F. graminearum infection. Using TargetP, a prediction of subcellular localization of
all the identified proteins based on their N-terminal amino acid
sequences was carried out [16]. Of the 41 proteins, 13 were
predicted to have specific subcellular localization. Four, 2, and
7 proteins were suggested to be located in the secretion pathway, mitochondria, and chloroplast, respectively. This implies
that chloroplasts are the organelles inside wheat cells mostly
affected by F. graminearum infection.
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Table 1. Expression level and identification of proteins responsive to F. graminearum infection in the spikes of Crystal
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Proteomics 2006, 6, 4599–4609
Table 1. Continued
*Y axis: normalized expression volume of the spot
*X axis: column1: 1 day H2O, 2: 1 day F. graminearum, 3: 2-days H2O, 4: 2-days F. graminearum, 5: 3-days H2O, 6: 3-days F. graminearum
§
for up- and down-regulated spots, maximum fold changes of expression volume averaged for 2 biological experiments and 3 technical
replicates for each experimental set
na: not applicable
{
: c: chloroplast; m: mitochondrion; s: secretory pathway; ,: any other location
3.4 DNA-damage inducible protein is glycosylated
after F. graminearum infection
Figure 4 shows that the DNA-damage inducible protein (A3)
is also a glycoprotein because it was stained with Pro-Q
Emerald, a glycoprotein-specific stain. Accessing the NetOGlyc 3.1 server to analyze the sequence of a corresponding
homologous rice protein (XP_464492), four threonines at
amino acids 346, 351, 360 and 361 (C-terminal end) were
identified as potential O-glycosylation sites within this protein. However, no potential N-glycosylation site could be
predicted for this protein.
3.5 F. graminearum proteins identified in the infected
spikelets
Six of the eight fungal proteins were identified as hypothetical
proteins in the NCBI database. Further annotation was performed by searching conserved domains within their sequen© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ces (Table 2). C1 carries the conserved domain pfam00724 for
flavin oxidoreductase. In E. coli, flavin oxidoreductase is a soluble enzyme which, under aerobic conditions and together
with NAD(P)H and flavins, generates superoxide radicals
selectively [24]. C2 carries cd00154, a conserved domain for Rab
GTPases. Rab GTPases are implicated in vesicle trafficking.
Different Rab GTPases are localized on the cytosolic face of
specific intracellular membranes, where they function as regulators in distinct steps of the membrane traffic pathway. In the
GTP-bound form, the Rab GTPases recruit specific sets of
effector proteins into membranes. Through their effectors, Rab
GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion [25].
C3 carries cd00946 that belongs to fructose-1,6-bisphosphate
(FBP) aldolase. This enzyme catalyses the zinc-dependent,
reversible aldol condensation of dihydroxyacetone phosphate
with glyceraldehyde-3-phosphate to form FBP. FBP aldolase is
homodimeric and used in gluconeogenesis and glycolysis [26].
C4 carries an uncharacterized conserved domain in bacteria.
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Proteomics 2006, 6, 4599–4609
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Figure 1. Sypro Ruby-stained
protein expression profile of
samples extracted from spikes
of Crystal that were harvested 1
and 2 days post inoculation with
F. graminearum and H2O. This is
a representative figure from
three technical and two biological replicates.
Figure 2. Sypro Ruby stained protein expression profile of sample extracted from spikes of Crystal wheat harvested 3 days post inoculation
with F. graminearum (a) or control (b). Labeled proteins were detected to be up-regulated or newly induced (A) or down-regulated (B) or
originate from the fungus (C) following F. graminearum infection. This is a representative figure from three technical and two biological
replicates.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Proteomics 2006, 6, 4599–4609
Figure 3. Enlarged inlets and expression
histograms showing examples of three
types of proteins that were differentially
expressed due to F. graminearum infection. A22, A8 and B2 are defined as an
induced, up-regulated, and down-regulated protein, respectively. Y axis: normalized expression volume of the protein; X axis: 1: 1 day H2O, 2: 1 day F. graminearum, 3: 2 days H2O, 4: 2 days
F. graminearum, 5: 3 days H2O, 6: 3 days
F. graminearum.
C5 is possibly a translation initiation factor because it carries
the conserved domain KOG3721, which belongs to the
translation initiation factor 5A. C6 was identified as an SOD
in the NCBI database, and the conserved domain search
reveals that it is a copper/zinc binding SOD. SOD catalyses
the conversion of superoxide radicals to H2O2 and molecular
oxygen. C7 carries pfam00254, a signature of the FKBP-type
peptidyl-prolyl cis-trans isomerase. It is a class of chaperone
related to trapping and refolding denatured proteins via
PTMs and affects protein turnover [27]. C8 is identified as
glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
which catalyzes the oxidative phosphorylation of glyceraldehydes-3-phosphate into 1, 3-biphosphoglycerate in the
glycolysis pathway [28].
4
Figure 4. Glycoprotein (GP) stain with the Pro-Q Emerald 488
and total protein (TP) stain with Sypro Ruby of proteins extracted
from 3-day post F. graminearum and H2O-inoculated spikelets of
Crystal. DNA-damage inducible protein (A3) was induced and
glycosylated following F. graminearum infection. Arrow points at
A3. Circles show that there is not a protein in the corresponding
position in either GP- or TP-stained gels of H2O control sample.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Discussion
We present the results of a proteomic analysis of wheat spikelets subjected to a compatible F. graminearum infection.
Using a fluorescence staining method, around 1380 proteins
from F. graminearum inoculated and controlled wheat spikelets were visualized on 2-D gels for identification and quanwww.proteomics-journal.com
Plant Proteomics
Proteomics 2006, 6, 4599–4609
4607
Table 2. Conserved domains of eight F. graminearum proteins identified by LC-MS/MS
Spot Accession Species
no. in NCBI
Conserved domain
CD
% of seq Score E-value
length aligned (bits)
Possible
function
C1
C2
C3
C4
C5
C6
C7
C8
pfam00724, Flavin oxidoreductase/NADH oxidase family
cd00154, Rab subfamily of small GTPases
cd00946, Fructose-1,6-bisphosphate (FBP) aldolases
COG3812, Uncharacterized protein conserved in bacteria
KOG3271, Translation initiation factor 5A
pfam00080, Copper/zinc superoxide dismutase
pfam00254, FKBP-type peptidyl-prolyl cis-trans isomerase
COG0057, Glyceraldehyde-3-phosphate dehydrogenase
335
165
345
193
156
152
95
335
Metabolism
Signal transduction
Glycolysis
Unknown
Translation initiation
Defense response
Protein folding
Glycolysis
EAA68107
XP_757798
EAA67336
EAA75300
EAA72434
EAA72418
EAA77739
EAA73952
F. graminearum
Ustilago maydis
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
F. graminearum
titative analyses of differentially regulated proteins in response to F. graminearum infection at anthesis. As a result,
we found 33 plant proteins that were responsive to F. graminearum infection and 8 fungal proteins from F. graminearum-infected spikelets. All the identified plant proteins
could be divided into two major groups based on their functions in relation to defense response and metabolism.
The first major group of proteins in response to F. graminearum infection are defense response proteins, including
those proteins with potential functions related to oxidative
burst pathway, signaling pathway, and PR-proteins. Three
proteins, ascorbate peroxidase (A7), glutathione transferase (A9), and osr40c1 (A17), have antioxidant function. This
suggests that there is a potential for mounting oxidative
burst for the purpose of defending invading fungus inside
wheat spike cells after initial infection of F. graminearum. In
a previous study, Zhou et al. [9] found that several antioxidant
proteins such as peroxiredoxins, GST, SOD, and dehydroascorbate reductase were up-regulated or induced 5 days
post inoculation with F. graminearum in the resistant wheat
line Ning7840. From this current study, we presented indirect evidence that a potential oxidative burst was induced by
F. graminearum and found that this defense activity happens
as early as the 1 day post inoculation because ascorbate peroxidase was shown to be up-regulated in the spikelets 1 day
post inoculation of F. graminearum. Osr40c1 was reported to
be responsive to salt tolerance and plays a role in the adaptative response of roots to a hyper-osmotic environment in rice
[29, 30]. Alternatively, due to the presence of fungal flavin
oxidoreductase, the pathogen could also generate radical
superoxide to attack the plant cells. All these detected antioxidant proteins were important to wheat cells for self protection against reactive oxygen species produced by themselves and fungus. Two proteins located in the signaling
pathway were found to be induced 3 days post F. graminearum inoculation. Ankyrin repeat protein (A2) is a regulator for both jasmonic acid (JA) and salicylic acid (SA) signaling pathways and mediates reciprocal inhibition of JA
responses by the SA signaling pathway [31, 32]. A15 was
identified as 12-oxo-phytodienoic acid reductase, an enzyme
of the biosynthetic pathway that converts linolenic acid to JA
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
98.8
100
100
86
93.6
94.1
100
99.1
243
278
521
125
61.5
194
101
460
7.00E-65
5.00E-76
4.00E-149
3.00E-30
8.00E-11
4.00E-51
3.00E-23
9.00E-131
[33]. Up-regulation of both ankyrin-repeat protein and 12oxo-phytodienoic acid reductase suggested that JA pathway is
most likely stimulated and SA pathway is inhibited during
F. graminearum infection. Three PR-proteins, b-glucanase (A4), chitinase (A11), and thaumatin-like protein (A19),
were detected to be induced or up-regulated due to F. graminearum infection. Chitinases and b-glucanases have a synergistic antifungal activity [34] and they also release molecules
that may act as elicitors [35]. Barwin, a wound-induced protein (A24) and a cold acclimation protein (A20) were also
detected to be up-regulated, suggesting that there are some
similarity between wheat responses to F. graminearum and
abiotic stresses such as wounding and low temperature.
The second major group of proteins detected to be
responsive to F. graminearum infection are involved in metabolism. Up-regulation of translation initiation factor (A18)
and transcription factor (A21) indicates that significant transcription changes were induced in wheat cells. Both a large
(A16) and a small (A23) RubisCO subunits were found to be
induced. However, their experimental molecular weights
were significantly lower than their theoretical values, thus
indicating an accelerated degradation of RubisCO following
F. graminearum infection. Down-regulation of RubisCO activase (B6) and GAPDH (B5) and degradation of RubisCO
suggest that photosynthesis was disrupted or at least
decreased after F. graminearum infection. This is supported
by the premature discoloration of wheat spike following
F. graminearum infection [1]. As expected, the down-regulation of vacuolar invertase (B1) strongly suggests that the
process of sucrose partitioning was affected in infected tissues, a clear impact of the disruption of photosynthesis.
After penetration of fungal mycelium in plant tissues,
F. graminearum has to acquire nitrogen and carbon from
wheat. Induction or up-regulation of cysteine synthase (A10),
tryptophan synthase (A8) and glutamate dehydrogenase (A13)
suggested that significant alteration of amino acid synthesis
and nitrogen metabolism were triggered by F. graminearum
infection in wheat. Solomon and Oliver [36] reported that the
content of nitrogen and most amino acids in the tomato leaves
increased during infection by Cladosporium fulvum. They also
found that cysteine and tryptophan were the only 2 of
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W. Zhou et al.
20 amino acids that were not detectable in tomato leaves [36].
It is most likely that wheat increases the synthesis of cysteine
and tryptophan to meet the both needs for its own synthesis of
PR-proteins and to compensate due to the sink created during
growth of fungal mycelia after F. graminearum infection. Four
proteins, two from wheat (A1 and B5) and two form F. graminearum (C3 and C8), were consecutive enzymes involved in
glycolysis. Both B5 and C8 were identified as GAPDH, but
were from wheat and F. graminearum, respectively. Phosphoglycerate kinase (A1) catalyzes the reversible reaction: ATP
1 3-phospho-D-glycerate ↔ ADP 1 3-phospho-D-glyceroyl
phosphate in the glycolysis process. GAPDH (B5 and C8):
catalyzes the reversible reaction: D-glyceraldehyde 3-phosphate 1 phosphate 1 NADP1 ↔ 3-phospho-D-glyceroyl
phosphate 1 NADPH in glycolysis. FBP aldolase (C3) catalyzes the reversible aldol condensation of glyceraldehyde
3-phosphate and dihydroxyacetone phosphate yielding FBP.
Up-regulation of phosphoglycerate kinase and down-regulation of GAPDH in wheat and detection of FBP aldolase and
GAPDH from F. graminearum suggested a possible connection of glycolysis between F. graminearum and wheat, in
which the fungus assimilates carbon from wheat. It might be
that phosphoglycerate kinase was stimulated and GAPDH
was inhibited in wheat cells by fungal growth because it
requires glyceraldehydes 3-phosphate as carbon source from
wheat. F. graminearum assimilates it and dihydroxyacetone
phosphate into fructose with its own aldolase. The obtained
fructose can be further converted into mannitol with mannitol
dehydrogenase by F. graminearum [37]. Mannitol is a common
storage carbon for most fungi and it can also serve as a
quencher of reactive oxygen species such as H2O2 of the plant
defense response, possibly aiding in pathogen colonization
[38]. It is reasonable that F. graminearum first uses SOD to
reduce the radical superoxide (O22) to form H2O2 and O2 and
then uses the mannitol to reduce H2O2.
The DNA-damage inducible protein (A3) was induced
and glycosylated after F. graminearum infection. Glycosylation is an important PTM of proteins in which oligosaccharides are attached to proteins by a variety of glycosidases and
glycosyltransferases [39]. Alterations in glycosylation profiles
are often useful indicators for the assessment of disease
states [12]. In the current study, direct detection of glycoproteins in gels with Pro-Q Emerald 488 dye, and subsequent
staining of all proteins with Sypro Ruby allowed us to directly
compare the expression and post-translation changes of differentially regulated proteins such as A3.
Crystal, the cultivar used in our study, was assessed to be
susceptible to F. graminearum based on phenotypic observations because it lacks the ability to inhibit the fungus
spreading from the inoculated spikelet to neighboring
spikelets. Resistant wheat lines that can inhibit or slow down
the fungus spread, and hence the disease, to adjacent spikelets, show the same initial infection pattern on the inoculated
spikelet as susceptible lines [5]. Our current study focused on
a compatible interaction between F. graminearum and wheat
because of the susceptibility of Crystal. Our results suggest
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 4599–4609
strongly an interaction between the fungus and wheat both
in the antioxidant and glycolysis pathways. The fungus can
overcome oxidative burst and obtain nutrition supply from
its wheat host successfully. However, even in a susceptible
cultivar, we were also able to detect the major components
for systemic acquired resistance such as production of antioxidant proteins, activation of JA pathway and up-regulation
of PR-proteins. The extent of the over-accumulation of these
proteins might be a determinant factor. Defense factors other
than those mentioned above may contribute to limit F. graminearum spread and possibly were not revealed in current
study. Alternatively, the magnitude of the plant response
might also be very important in a resistant line. A future
study will consider the response level of genes identified in
this study in both resistant and susceptible lines.
In summary, the present proteomic investigation of
wheat spikelets susceptible to F. graminearum revealed a
complex cellular network in the wheat cells in response to
the fungus infection. The network covers oxidative burst, JA
and SA signaling pathways, generation of PR-proteins, protein synthesis, photosynthesis and other metabolic pathways. Glycosylation of a DNA-damage inducible protein was
also detected. Subcellular localization of proteins in response
to F. graminearum infection revealed that the protein complement of chloroplasts is one of the organelles inside wheat
cells mostly affected by F. graminearum. Our research also
revealed that F. graminearum directly interacts with wheat in
two pathways: antioxidant and glycolysis, in which the
pathogen overcomes reactive oxygen species and obtains
carbon from wheat, respectively. The availability of the complete genome of F. graminearum assisted us to identify fungal proteins in infected spikelets. It is noteworthy that only
10 of the 33 identified plant proteins were directly identified
as originating from T. aestivum showing the importance to
continue genomic and proteomic research in this very
important crop species. Availability of the complete wheat
genome in the future would be very beneficial for identification of polypeptides identified in proteomic projects.
We thank Dr. Allis Chien at the Stanford University Mass
Spectrometry Center for protein identification with LC-MS/MS.
The funding for this project was partially provided by Alberta
Agricultural Research Institute.
5
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