Journal of Experimental Botany, Vol. 59, No. 9, pp. 2371–2378, 2008
doi:10.1093/jxb/ern103 Advance Access publication 7 May, 2008
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Transgenic wheat expressing a barley class II chitinase
gene has enhanced resistance against Fusarium
Sanghyun Shin1, Caroline A. Mackintosh1,*, Janet Lewis1,†, Shane J. Heinen1, Lorien Radmer1,
Ruth Dill-Macky2, Gerald D. Baldridge2, Richard J. Zeyen2and Gary J. Muehlbauer1,‡
1Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford
Circle, St Paul, MN 55108, USA
2Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St Paul,
MN 55108, USA
Received 25 November 2007; Revised 13 March 2008; Accepted 14 March 2008
Fusarium head blight (FHB; scab), primarily caused by
Fusarium graminearum, is a devastating disease of
wheat worldwide. FHB causes yield reductions and
contamination of grains with trichothecene mycotox-
ins such as deoxynivalenol (DON). The genetic varia-
tion in existing wheat germplasm pools for FHB
resistance is low and may not provide sufficient
resistance to develop cultivars through traditional
breeding approaches. Thus, genetic engineering pro-
vides an additional approach to enhance FHB resis-
tance. The objectives of this study were to develop
transgenic wheat expressing a barley class II chitinase
and to test the transgenic lines against F. graminea-
rum infection under greenhouse and field conditions.
A barley class II chitinase gene was introduced into
the spring wheat cultivar, Bobwhite, by biolistic bom-
bardment. Seven transgenic lines were identified that
expressed the chitinase transgene and exhibited en-
hanced Type II resistance in the greenhouse evalua-
tions. These seven transgenic lines were tested under
field conditions for percentage FHB severity, percent-
age visually scabby kernels (VSK), and DON accumu-
lation. Two lines (C8 and C17) that exhibited high
chitinase protein levels also showed reduced FHB
severity and VSK compared to Bobwhite. One of the
lines (C8) also exhibited reduced DON concentration
compared with Bobwhite. These results showed that
transgenic wheat expressing a barley class II chitinase
exhibited enhanced resistance against F. graminearum
in greenhouse and field conditions.
Key words: Chitinase, Fusarium graminearum, Fusarium head
blight, transformation, wheat.
Fusarium head blight (FHB; scab), primarily caused by
Fusarium graminearum Schwabe (teleomorph Gibberella
zeae (Schwein.) Petch: synonym¼G. saubinetti), is a seri-
ous disease of wheat and other small grains in hot and
humid regions around the world. Between 1998 and 2000,
FHB caused an estimated 2.7 billion dollar economic loss
in the Midwestern United States (Nganje et al., 2004).
FHB reduces yield through discoloured and shrivelled
‘tombstone’ kernels. Grain quality is also reduced due to
accumulation of trichothecene mycotoxins such as deoxy-
nivalenol (DON), and the estrogenic zearalenone (McMullen
et al., 1997).
Host resistance in wheat has been considered the most
practical and effective means of FHB disease control;
however, wheat breeding programmes have been limited
by a lack of effective resistance genes (Bai and Shaner,
1996). Two major types of FHB resistance have been
classified. Type I resistance is a reduction in initial
infection, and Type II resistance is reduced spread of
* Present address: University of Saint Mary, 4100 S. 4th Street Leavenworth, Kansas 66048, USA.
yPresent address: Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824, USA.
zTo whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Abbreviations: FHB, Fusarium head blight; GUS, b-glucuronidase; VSK, visually scabby kernels; DON, deoxynivalenol.
ª 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
disease symptoms in the spike (Schroeder and Christen-
sen, 1963). Quantitative trait loci (QTL) that confer Type I
and Type II resistance have been identified (Waldron
et al., 1999; Buerstmayr et al., 2003). To increase FHB
resistance, wheat-breeding programmes select for both
Type I and Type II resistance (Rudd et al., 2001).
However, wheat germplasm sources identified to date
exhibit partial resistance. Thus, genetic engineering
provides an additional approach to increase the level of
FHB resistance in wheat.
Several classes of genes have been used in a genetic
engineering approach to develop resistance in wheat to
fungal pathogens. One group of genes, referred to as
defence response genes encode proteins such as: b-1,3-
glucanases, chitinases, thaumatin-like proteins (tlps),
ribosome-inactivating protein (RIPs), and thionins. The
defence response genes function in a variety of ways to
inhibit fungal infection and expression of these genes in
transgenic plants has been shown to enhance fungal
resistance (Muehlbauer and Bushnell, 2003). Expressing
defence response genes in transgenic wheat resulted in
enhanced resistance to the powdery mildew (Bliffeld
et al., 1999; Oldach et al., 2001; Bieri et al., 2003) and
leaf rust pathogens (Oldach et al., 2001). With respect to
FHB, wheat lines expressing b-1,3-glucanase, thaumatin-
like protein1 (tlp-1), ribosome-inactivating protein (RIP),
a-1-purothionin, and Arabidopsis NPR1 (AtNPR1) trans-
genes exhibited enhanced resistance against F. graminea-
rum in greenhouse and/or field trials (Chen et al., 1999;
Makandar et al., 2006; Balconi et al., 2007; Mackintosh
et al., 2007).
Chitinases (EC 188.8.131.52) break bonds between the C1
and C4 of two consecutive N-acetylglucosamines of
chitin, which is a main component of the cell wall in
fungi. Plant chitinases are characterized as pathogenesis-
related proteins and are classified into seven classes (I–
VII) based on their primary structures (Flach et al., 1992;
Collinge et al., 1993). Chitinase genes are up-regulated
during early infection of wheat and barley spikes by F.
graminearum (Pritsch et al., 2000, 2001; Li et al., 2001;
Kang and Buchenauer, 2002; Kong et al., 2005; Boddu
et al., 2006, 2007; Bernardo et al., 2007; Golkari et al.,
2007). Expression of a rice chitinase transgene in rice,
Italian ryegrass, and grapevine resulted in enhanced
resistance to the rice blast, crown rust, and powdery
mildew pathogens, respectively (Nishizawa et al., 1999;
Yamamoto et al., 2000; Takahashi et al., 2005). Trans-
genic wheat lines carrying an overexpressed wheat
chitinase and b-1,3-glucanase combination showed partial
resistance to FHB in greenhouse evaluations; however, the
lines did not exhibit improved resistance under field
conditions (Anand et al., 2003). Wheat plants constitu-
tively expressing a barley class II chitinase transgene also
showed resistance against the powdery mildew and leaf
rust pathogens (Bliffeld et al., 1999; Oldach et al., 2001).
The efficacy of transgenic wheat expressing a barley class
II chitinase against the powdery mildew and leaf rust
fungal pathogens made it an obvious choice to test against
The objectives of this study were to develop transgenic
wheat carrying a barley class II chitinase transgene and
evaluate these lines during F. graminearum infection for
resistance in the greenhouse and in the field. Seven
transgenic wheat lines that exhibited enhanced Type II
FHB resistance in the greenhouse have been identified.
Two of these lines exhibited high levels of chitinase
protein and enhanced FHB resistance in the field.
Materials and methods
The spring wheat cultivars Alsen, 2375, Roblin, Sumai 3, Wheaton,
and Bobwhite were used for the experiments. Wheaton and Roblin
are hard red spring wheat cultivars that are highly susceptible to
FHB; Bobwhite was used for the transformations and is slightly less
susceptible than Wheaton; 2375 is moderately susceptible to FHB;
Alsen exhibits Type II resistance and is moderately resistant to
FHB; Sumai 3 is a Chinese cultivar exhibiting Type I and Type II
resistance (Bai and Shaner, 1996).
Plant transformation plasmids
pAHC25: The pAHC25 plasmid (Christensen and Quail, 1996; gift
from Dr Peter Quail, USDA-ARS, Albany, CA) contains the uidA
and bar genes driven by the maize ubiquitin promoter. The uidA
gene encodes the b-glucuronidase (GUS) enzyme and the bar gene
encodes the phosphinothricin acetyltransferase (PAT) enzyme.
PAT activity confers resistance to phosphinothricin-containing
pAHCBarChit: The 998 bp barley seed class II chitinase cDNA
(GenBank accession number M62904; Leah et al., 1991; a gift from
Dr John Mundy, Carlsberg Research Laboratory, Copenhagen,
Denmark) was cloned into the BamHI site of pAHC17. The barley
class II chitinase cDNA sequence contains an 801 bp open reading
frame, beginning with the first ATG initiation codon at nucleotide
position 61 and ending with a TAA termination codon at position
862. The pAHC17 plasmid (Christenson and Quail, 1996; a gift
from Dr Peter Quail, USDA-ARS, Albany, CA) contains the maize
ubiquitin promoter/exon/intron (UBI-1) sequence and the Agro-
bacterium tumefaciens nopaline synthase 3,- end sequence. The
orientation within the plasmid and open reading frame integrity of
the barley chitinase gene were confirmed by sequencing.
The spring wheat cultivar Bobwhite was used as the host for
transformation. All aspects of the transformation protocols in-
cluding particle gun bombardment of embryos, tissue culture
selection and plant regeneration was conducted according to
Mackintosh et al. (2006). A 1:1 ratio of pAHC25 and pAHC-
BarChit, 5 lg each, were cotransformed into Bobwhite. During the
selection and regeneration process, the identity of the callus and
shoots derived from each embryo was maintained. To ensure that
each line was independent, only a single plant expressing the
transgene from each embryo was advanced for testing.
2372 Shin et al.
RNA isolation and transcript analysis
Total RNA was isolated from 100 mg leaf tissue with TRIZOL?
reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s
instructions. Reverse transcription polymerase chain reactions (RT-
PCR) were performed with 1 lg of total RNA using the
Superscript? III One-step RT-PCR kit (Invitrogen, Carlsbad, CA).
For the chitinase transgene and actin control, the reverse transcrip-
tase reactions were performed in a thermal cycler at 55 ?C for
30 min, followed by 35 cycles of amplification (denature at 94 ?C for
15 s, annealing at 60 ?C for 1 min and extension at 68 ?C for 1 min)
and final extension of 68 ?C for 5 min. The primer pair (5#-
GATGCATATACATGATGGCATATGCAG-3#, and 5#-GTCCA-
TAGTTGTAGTTGTGGGAGAG-3#) was used for amplification of
the chitinase mRNA. The expected size for the amplified products
from the chitinase mRNA was 742 bp. The primer pair (5#-
GCCACACTGTTCCAATCTATGA-3# and 5#-TGATGGAATTG-
TATGTCGCTTC-3#) was used for amplification of the wheat actin
control gene. The expected band size for the wheat actin gene was
369 bp. Sequence analysis of the chitinase and actin RT-PCR
products confirmed that the correct transcripts had been amplified.
Southern blot analysis
Southern blot analysis was performed according to de la Pen ˜a et al.
(1996). A radiolabelled portion of the ubiquitin promoter and
chitinase transgene was used as a probe for the hybridizations. The
probe was derived from the PCR primers used in the RT-PCR
reactions, resulting in a 742 bp probe (Fig. 1).
Greenhouse screening of transgenic lines against
F. graminearum infection
Seed from each wheat genotype were planted into Metro-Mix 200
growth medium (The Scotts Company, Marysville, OH) in 6#
square plastic pots in a greenhouse. Twenty seeds were planted for
each line with each pot containing five seeds. Plants were fertilized
with one teaspoon of Osmocote (14-14-14 N-P-K, Scotts Company,
Marysville, OH) fertilizer per pot at the 3-leaf stage. At anthesis,
a single central floret of the spikelet of the main stem was
inoculated with 10 ll of a macro-conidial suspension (100 000
conidia ml?1) of isolate Butte86Ada-11 (Evans et al., 2000) of F.
graminearum. Inoculated spikes were bagged in plastic and the
plants were placed in a dew chamber for 72 h and subsequently
moved back to the greenhouse. The number of visually symptom-
atic spikelets, including the inoculated spikelet on each plant, were
counted 20 d after inoculation (dai). The disease severity was
determined as the percentage of infected spikelets on the inoculated
spikes with visually detectable disease symptoms. In each green-
house screen, non-transformed Bobwhite, Wheaton, and Sumai 3
were used. For statistical analysis, Student’s t tests were used to
compare each transgenic line to the parental Bobwhite controls. All
analysis was performed with Microsoft Excel Version 2003 (Micro-
soft Corporation, Redmond, WA).
Field screening of transgenic lines against F. graminearum
Transgenic wheat lines were evaluated in the field against F.
graminearum. Seed for each transgenic line was derived from
selfing plants that exhibited expression of the chitinase transgene. It
is possible that the transgene was still segregating in the lines tested
in the field. Bobwhite, Alsen, 2375, Norm, Roblin, and Wheaton
were included in the experiment as disease checks. An additional
non-inoculated treatment of Wheaton was also used to establish the
background level of inoculum. Two experiments were conducted:
one during the summer of 2005 at the University of Minnesota
Agricultural Experiment Station in Crookston, MN and another in
the summer of 2007 at the University of Minnesota Agricultural
Experiment Station (UMore Park) in Rosemount, MN. T6and T8
lines were used for the 2005 and 2007 field tests, respectively. The
experimental design was a randomized complete block with four
replications. Each genotype was planted in two-row plots; rows
were 2.4 m long and were spaced 0.3 m apart. Within each row,
3.3 g m?1of seed was sown.
The inoculum consisted of a mixture of 50 isolates of F.
graminearum in 2005 and 41 isolates in 2007. The isolates were
obtained from naturally FHB-infected commercial wheat and barley
fields in Minnesota from 2004 and 2006. The plots were inoculated
at anthesis and then 3 d later. Each row was inoculated with 33 ml
m?1of inoculum mixture (13105macroconidia ml?1). Inoculum
was applied using a CO2-powered backpack sprayer fitted with
a TeeJet?(Spraying Systems Co., Wheaton, IL) SS80015 flat-fan
nozzle that was operated at a pressure of 276 kPa.
FHB disease severity was evaluated visually 21 dai. Twenty
spikes from primary tillers were arbitrarily selected per plot and
rated for disease severity. Disease severity was measured as the
percentage of symptomatic spikelets per spike. After harvest, the
grain was assessed for the percentage of visually scabby kernels
(VSK) on a hand-cleaned 50 g sample. VSK values were assigned
based on standards with a known percentage of scabby kernels
(Jones and Mirocha, 1999). After VSK analysis, the samples were
ground for 2 min with a Stein Laboratory Mill and deoxynivale-
nol (DON) concentration was determined using gas chromatogra-
phy and mass spectrometry with the procedure described in
Mirocha et al. (1998) with slight modifications. For statistical
analysis, Student’s t tests were used to compare each transgenic
line to the parental Bobwhite controls. All analysis was performed
with Microsoft Excel Version 2003 (Microsoft Corporation,
Western blot analysis
Protein was extracted by grinding spikes at anthesis in extraction
buffer (50 mM NaH2PO4, pH 6.8, 100 mM PMSF) and cell debris
was removed by micro-centrifugation. Total protein concentration
was determined using Bio-Rad reagent (Bio-Rad, Hercules, CA)
with bovine serum albumin as a standard. Protein extracts (10 lg)
were separated by SDS-polyacrylamide electrophoresis (12% acryl-
amide) and transferred to PVDF transfer membrane (Amersham
Biosciences, Piscataway, NJ). Affinity-purified polyclonal chitinase
antibodies were developed from two peptides (SRAQFDRMLLHR-
NDGAC and CGKRYYGRGPIQLSHNYNY) from the barley chiti-
nase protein. The polyclonal chitinase antibody was developed by
Quality Controlled Biochemicals, Inc., Hopkinton, MA. The blots were
incubated with a 1:1000 dilution of the chitinase polyclonal antibody.
Fig. 1. The pAHCBarChit plasmid containing the barley class II
chitinase transgene was used for wheat transformation. The arrow
indicates the region amplified in the RT-PCR assays and the region
used for the Southern blot probe. The ubiquitin 1 promoter and intron is
from the maize ubiquitin gene and the T nos termination sequence is
from the nopaline synthase gene from Agrobacterium tumefaciens.
Transgenic wheat and scab resistance 2373
Chitinase protein was visualized using an ECL Western Blotting
Reagent Pack (rabbit) (Amersham Biosciences, Piscataway, NJ).
Generation of transgenic wheat plants
The pAHCBarChit plasmid (Fig. 1), which included the
barley chitinase gene driven by the maize ubiquitin
promoter, and pAHC25 which carries the bar gene as
a selectable marker and the uidA (GUS) as a reporter
gene, were co-bombarded into the wheat cultivar Bob-
white. Plants were selected on bialaphos and only plants
that exhibited GUS expression were regenerated. To
identify plants that exhibited expression of the barley
chitinase transgene, RT-PCR analysis was conducted on
the T0plants. Sixteen transgenic wheat plants expressing
the chitinase transgene were identified. The 16 wheat
plants were advanced to T2lines by selfing T1plants that
expressed the transgene based on the RT-PCR assay.
Greenhouse screening of transgenic plants for FHB
FHB resistance was evaluated in the greenhouse of these
16 transgenic wheat lines that exhibited barley chitinase
transgene expression. Each line was examined in at least
three generations spanning the T2to T4or T2to T5and
each line was examined in at least three separate disease
screens. Based on an RT-PCR assay for the chitinase
transgene, advanced generations were obtained through
selfing plants that expressed the barley chitinase trans-
gene. Thus, in each disease screen it is still possible that
the lines were segregating null, homozygous or hemi-
zygous for the transgene. For each screen, 20 plants of
each line were grown and the spikes were inoculated with
F. graminearum. The central spiklelet in primary spikes
were point inoculated and examined visually for the
spread of disease 20 dai. RT-PCR was used to assay each
plant in the transgenic lines for chitinase transgene
expression. Based on the RT-PCR assays, only those
plants expressing the chitinase transgene were used to
evaluate the efficacy of chitinase against F. graminearum.
FHB severity in the transgenic lines expressing the
chitinase transgene was compared to the severity observed
in the non-transgenic Bobwhite parent.
In the greenhouse experiments, seven transgenic lines
(C3, C4, C6, C8, C12, C15, and C17) had significantly
reduced FHB severity in at least two FHB disease screens
when compared to the Bobwhite control (P <0.05; Table 1).
No significant difference was observed in the remaining
nine transgenic lines in the FHB disease screens (data not
shown). The transgenic C8 and C12 lines had significantly
reduced FHB severity compared to the untransformed
Bobwhite controls in three greenhouse screens (P <0.05).
The average reduction of disease severity across all the
trials in C8 and C12 was 46% and 58%, respectively. The
remaining lines C3, C4, C6, C15, and C17 lines exhibited
reduced severity in two disease screens. C3, C4, C6, C15,
and C17 exhibited an average reduction in severity of
40%, 30%, 36%, 52%, and 31%, respectively. Overall, all
seven lines showed a similar level of resistance to FHB.
Molecular analysis of transgenic plants
To verify that the seven lines that exhibited enhanced
resistance in the greenhouse screens were independently
derived and transgenic, DNA gel blot analysis was
conducted. Genomic DNA from the seven lines was
digested with NcoI. Based on the plasmid map for
Table 1. Percentage of Fusarium head blight severity in transgenic wheat carrying a barley chitinase in greenhouse evaluations
Numbers in parenthesis represent the number of plants expressing the chitinase transgene, based on the RT-PCR assay, in the screen.
Autumn 2002 Winter 2003 Spring 2003Autumn 2003 Autumn 2003Spring 2004 Spring 2004
– 47* (13)
– 57 (16)
60 (22)68 (46)60 (22)
aC3, C4, C6, C8, C12, C15, C17 are transgenic wheat lines carrying the barley chitinase. Bobwhite was the untransformed control, Wheaton was
the FHB susceptible check, and Sumai 3 was the FHB resistant check.
bGreenhouse testing in autumn 2002, winter 2003, spring 2003, autumn 2003, autumn 2003, spring 2004, and spring 2004 corresponded to T2, T2,
T3, T3, T4, T4, and T5for the transgenic lines, respectively. FHB severity was measured as the percentage of infected spiklets per head 20 d after
inoculation. – Indicates that this line was not examined in this screen. *, **, *** indicates significance at the 0.05, 0.01, and 0.001 level, respectively,
compared to Bobwhite (Student’s t test).
2374 Shin et al.
pAHBarChit this will result in 1.1 kb fragment. A portion
of the ubiquitin promoter and chitinase transgene was
used as a probe (Fig. 1). The probe hybridized weakly to
the Bobwhite control; however, each line exhibited an
approximately 1.1 kb fragment and each of the lines
exhibited a different banding pattern, indicating that the
seven lines were transgenic and resulted from independent
transformation events (Fig. 2).
To examine transgene expression in seven lines (C3,
C4, C6, C8, C12, C15, and C17) that exhibited resistance
against F. graminearum in the greenhouse screens, RT-
PCR and western blot analysis were conducted. Figure 3
is an example of the RT-PCR analysis of these seven
lines. Western blot analysis of protein expression was also
conducted. Protein from spikes was isolated at anthesis
from transgenic lines C3, C4, C6, C8, C12, C15, and C17
and the blots were incubated with antibody for the barley
chitinase protein. In the western blot shown in Fig. 4, the
cross-reaction of the chitinase antibody with protein from
Bobwhite and all the transgenic lines was observed. Three
transgenic lines (C8, C15, and C17) had a clearly higher
level of chitinase protein (Fig. 4). Similar results were
observed in other western blots prepared with leaf and
spike protein. These results indicate that C8, C15, and
C17 were the only lines that exhibited a high level of
Field disease screening of transgenic plants for FHB
To examine the efficacy of the wheat transgenic lines
expressing the chitinase transgene in providing improved
levels of resistance to F. graminearum, field evaluations
of seven lines (C3, C4, C6, C8, C12, C15, and C17) were
conducted in the summers of 2005 and 2007. FHB
severity, percentage of visually scabby kernels (VSK),
and DON concentrations on these seven lines and the non-
transgenic Bobwhite control were assessed (Table 2). In
the 2005 and 2007 field tests, the C17 line exhibited
significant reduction in percent FHB severity and VSK
compared to Bobwhite. The C8 line showed significant
reduction in percentage FHB severity, percentage VSK,
and in DON concentration. The disease severity in the C3,
C4, C6, C12, and C15 transgenic lines were either similar
to or higher than the non-transgenic Bobwhite control.
FHB is a serious disease of wheat and has resulted in
significant economic losses around the world. Available
FHB resistance in wheat is inherited in a quantitative
manner and is partial. Several QTL in wheat have been
identified that confer Type I and II resistance, with the
largest QTL explaining variation for Type II resistance
located on chromosome 3BS (Waldron et al., 1999;
Anderson et al., 2001; Buerstmayr et al., 2003). Multiple
studies have shown the induction of a large set of defence
Fig. 2. Southern blot analysis of seven transgenic wheat plants carrying
a barley chitinase. Genomic DNA from Bobwhite parent and transgenic
lines were digested with NcoI, and hybridized with a probe that bridges
the ubiquitin promoter and the chitinase transgene junction. The arrow
indicates the expected size of the 1.1 kb hybridizing fragment from
a NcoI digestion of the plasmid.
Fig. 3. RT-PCR analysis of transgenic wheat plants carrying a barley
chitinase gene. The expected size of the chitinase transgene fragment
was 742 bp. The wheat actin gene was used as a positive control and it
exhibited the expected size of 369 bp.
Transgenic wheat and scab resistance 2375
response genes in wheat following F. graminearum
infection (Pritsch et al., 2000, 2001; Li et al., 2001; Kang
and Buchenauer, 2002; Kong et al., 2005; Hill-Ambroz
et al., 2006; Bernardo et al., 2007; Golkari et al., 2007).
Chitinase, one of the defence response genes identified in
these studies, limits fungal growth by degrading the major
structural polysaccharide of fungal cell walls (Leah et al.,
1991). It has been proposed that overexpression of
a chitinase transgene protein may function to provide
fungal pathogen resistance on both direct and indirect
levels. On the direct level it degrades chitin of growing
hyphae, whereas on the indirect level it results in the
release of chitin oligomers which can act as elicitors of
plant defence mechanisms (Collinge et al., 1993). In this
report, it is shown that transgenic wheat expressing
a barley class II chitinase gene enhances resistance against
F. graminearum under greenhouse and field conditions.
To date, complete resistance against fungal pathogens
has not been achieved by the expression of single genes
encoding defence response genes. Expression of chitinase
transgenes of different origins resulted in enhanced
resistance in rice (Nishizawa et al., 1999), Italian ryegrass
(Takahashi et al., 2005), and grapevine (Yamamoto et al.,
2000) to Magnaporthe grisea, Puccinia coronata, and
Uncinula necator, respectively. Moreover, expression of
a barley chitinase gene in transgenic wheat resulted in
enhanced resistance to infection by Erysiphe graminis,
Blumeria graminis, and Pucinia recondita (Bliffeld et al.,
1999; Oldach et al., 2001; Bieri et al., 2003). Chen et al.
(1999) showed that expression of a rice thaumatin like
protein-1 (tlp-1) transgene in wheat resulted in enhanced
FHB disease during the early stages of disease progression
in the greenhouse, indicating a delay in FHB develop-
ment. A transgenic wheat line carrying a chitinase trans-
gene did not enhance FHB resistance compared to the
(Anand et al., 2003). However, the chitinase transgene
exhibited a low level of expression and was probably
silenced in the tested generation. Another wheat line
carrying a chitinase and b-1,3-glucanase exhibited delayed
susceptibility compared with non-transgenic controls in
greenhouse screens, but this line did not exhibit delayed
susceptibility in field screens (Anand et al., 2003).
Balconi et al. (2007) showed that transgenic wheat plants
expressing a maize RIP gene reduced the FHB disease
symptoms 14 dai. These authors only detected enhanced
FHB resistance during the early stages of disease pro-
gression in the greenhouse. In contrast to these reports,
FHB resistance was detected during the late stages of
disease progression (i.e. 20 and 21 dai for the greenhouse
and field, respectively). Seven lines were identified
expressing a barley chitinase transgene that exhibited
enhanced FHB resistance compared to the non-transgenic
Bobwhite control in greenhouse screens. Seven transgenic
lines in field trials were evaluated, and two lines were
identified that exhibited improved resistance against F.
graminearum in the field trials. Thus, our results show
that expressing defence response genes in transgenic
wheat can result in enhanced resistance against F.
graminearum. Consistent with these results, Mackintosh
et al. (2007) also showed that expression of the defence
response genes a-1-purothionin, tlp-1, and b-1,3-gluca-
nase in transgenic wheat exhibited resistance against F.
graminearum in the greenhouse and field trials.
Improved resistance was not detected in each of the
seven transgenic lines in every greenhouse screen. Lines
providing enhanced resistance were designated as those
that exhibited resistance in at least two of the greenhouse
screens. The lack of consistency in the disease screens is
probably due to the variability inherent in FHB disease
screens, which was also observed previously (Mackintosh
et al., 2007). Therefore, to detect transgenic wheat lines
Table 2. Percentage of Fusarium head blight (FHB) severity,
visual scabby kernels (VSK), and deoxynivalenol (DON)
concentration in transgenic wheat carrying a barley chitinase
evaluated in the field in 2005 and 2007
aC3, C4, C6, C8, C12, C15, C17 are transgenic wheat lines carrying
the barley chitinase transgene. T6and T8plants were evaluated in 2005
and 2007, respectively. Bobwhite was the untransformed control,
Wheaton and Roblin are FHB susceptible checks, and Alsen and Sumai
3 are a FHB resistant check, and 2375 is a moderately resistant check.
The non-inoculated treatment of Wheaton (non) was used to establish
the background level of inoculum.
bppm, parts per million.
cValues presented are the means of eight replicates (four replicates
tested in each 2005 and 2007). *, **, *** indicates significance at the
0.05, 0.01, and 0.001 level, respectively, compared to Bobwhite
(Student’s t test).
Fig. 4. Western blot analysis of transgenic wheat plants carrying
a barley chitinase gene. Total protein (10 lg) extracted from spikes of
transgenic lines was subjected to SDS-PAGE analyses. Molecular
markers indicated the protein to be the expected 26 kDa size.
2376 Shin et al.
carrying enhanced levels of FHB resistance, our results
demonstrate the importance of conducting multiple tests.
The transgenic lines that exhibited resistance in the
greenhouse did not all exhibit resistance in the field. In the
greenhouse, the spikes were point-inoculated and Type II
resistance was evaluated, whereas in the field the spikes
were spray-inoculated and disease severity, VSK, and
DON concentration were evaluated. The transgenic wheat
lines (C3, C4, C6, C12, and C15), which showed
enhanced Type II resistance in the greenhouse evalua-
tions, did not display detectable resistance in the field.
However, the transgenic wheat lines C8 and C17 did
show resistance in the field, and reduced the average
disease severity compared to Bobwhite by 39%. Yield and
grain quality reductions from FHB are due to fungal
damage to kernels, and contamination of grain by DON.
In our study, the C8 transgenic line also showed reduced
VSK and DON concentration in harvested grain, whereas
the C17 line exhibited reduced VSK. These results are
probably due to the chitinase transgene delaying the onset
of the FHB disease and thus reducing the colonization of
the developing wheat kernels and the production of
Chitinase protein levels in spikes of C8 and C17 were
correlated with the field results. In our study, the C8 and
C17 lines exhibited a high level of chitinase protein and
they were the only lines that exhibited FHB resistance in
the field. In contrast, the C15 line exhibited an increase in
chitinase protein compared to Bobwhite. However, for
unknown reasons this line did not result in enhanced
resistance against F. graminearum in the field. It is
possible that the inherent variation in FHB screens
resulted in the C15 line not showing a difference when
compared to Bobwhite. The chitinase protein levels in the
C3, C4, C6, and C12 transgenic wheat lines were
observable but indistinguishable from Bobwhite and
exhibited enhanced FHB resistance only in the green-
house. Interestingly, the C12 line exhibited the highest
level of resistance in the greenhouse and a low level of
chitinase protein, but did not show enhanced resistance in
the field. Balconi et al. (2007) showed that reduced FHB
symptoms in transgenic wheat lines carrying maize RIP
gene did not depend on the level of RIP protein. However,
Takahashi et al. (2005) showed that transgenic ryegrass
plants with a higher level of chitinase mRNA accumula-
tion and activity tended to have higher resistance to crown
rust disease (Puccinia coronata). Except for the anomaly
observed in C15, our results indicate that increased
chitinase protein is sufficient to enhance host resistance to
FHB in field-grown plants.
Wheat germplasm pools lack sufficient resistance to
develop FHB-resistant varieties. Sumai 3 is widely used
as a source of Type I and Type II resistance that limits
initial infection and disease spread, respectively. QTL
mapping in Sumai 3 and Sumai 3 derivatives have
identified the location of Type I and Type II resistance
and shown that Type II resistance is the major form of
FHB resistance (Anderson et al., 2001; Buerstmayr et al.,
2003). However, the level of Sumai 3-derived resistance is
insufficient for cultivars in severe FHB epidemics. Thus,
the transgenic lines presented here may provide a potential
wheat germplasm source for enhanced FHB resistance.
We are grateful to Dr John Mundy of the Carlsberg Research
Laboratory, Copenhagen, Denmark for providing the barley
chitinase cDNA. We would like to thank Dr Peter Quail (USDA-
ARS, Plant Gene Expression Center, Albany, CA) for providing the
pAHC25 and pAHC17 plasmids. We are indebted to Abigail Cole,
Sarah Jutila, Alissa Cyrus, Karen J Wennberg, Amar M Elakkad,
and Yanhong Dong for excellent technical assistance. Sanghyun
Shin was supported by the Korean Research Foundation (KRF-
2005-000-10035). This project was supported by funds from the
USDA-ARS US Wheat and Barley Scab Initiative, the Minnesota
Small Grains Initiative, and Minnesota Wheat Research and
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