Virus-Induced Gene Silencing-Based Functional
Characterization of Genes Associated with
Powdery Mildew Resistance in Barley1
Ingo Hein, Maria Barciszewska-Pacak, Katarina Hrubikova2
, Sandie Williamson, Malene Dinesen,
Ida E. Soenderby, Suresh Sundar, Artur Jarmolowski, Ken Shirasu, and Christophe Lacomme*
Programme of Genome Dynamics (I.H., S.W., S.S.) and Programme of Cell-to-Cell Communication
(M.B.-P., K.H., C.L.), Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom;
Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, 60–371 Poznan, Poland
(M.B.-P., A.J.); Danish Institute of Agricultural Sciences, Biotechnology Group, DK–1871 Frederiksberg C,
Copenhagen, Denmark (M.D.); Denmark Plant Biochemistry Laboratory, Department of Plant Biology,
The Royal Veterinary and Agricultural University, DK–1871 Frederiksberg C, Copenhagen, Denmark
(I.E.S.); and The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom (K.S.)
We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization
of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially,
barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its
replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching
was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular
level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based
functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene
silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could
be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly
developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew
strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was
observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required
component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes
involved in pathogen resistance in barley.
One of the best-studied cereal pathosystems for
investigating the genetic and molecular bases of monocotyledonous
plant-pathogen interactions is the association
between the obligate biotrophic powdery
mildew fungus Blumeria graminis f. sp. hordei (Bgh)
and its natural host barley (Hordeum vulgare). Powdery
mildew is one of the most important and devastating
diseases of barley worldwide. Genetic resistance in
barley to Bgh can be either race specific or nonrace
specific. Race-specific resistance is conditioned by the
interactions of resistance (R) gene products in the host,
such as those encoded by the complex Mla locus on
chromosome 1H, and the products of cognate Avr
genes in races of the fungus (Jørgensen, 1992).
Mutational analysis and map-based cloning in barley
have identified genes that are necessary to establish
Mla-mediated resistance to Bgh. This approach
identified RAR1, a small zinc-binding protein with
two highly similar domains, CHORD-I and -II (Cysand
His-rich domain), which is conserved in almost all
eukaryotes except for yeasts and metazoans. In the
latter species, equivalent proteins differ from plant
homologs by carrying an additional C-terminal domain,
the CS motif, with homology to the Saccharomyces
cerevisiae protein SGT1 (for suppressor of G-two allele of
Skp1; Shirasu et al., 1999). It has been shown that
RAR1 interacts with SGT1 protein both in yeast twohybrid
assays and in planta. In plants, vertebrates, and
yeast, SGT1 associates with SCF (Skp1-Cullin-F-box)type
E3 ubiquitin-ligase complexes potentially linking
disease resistance signaling cascades to ubiquitinationdependent
processes (Azevedo et al., 2002; Liu et al.,
2004). The CS domain, a central region of human SGT1,
exhibits a tertiary structure that is related to the
cochaperone p23 and binds to cytosolic heat shock
protein 90 (HSP90; Lee et al., 2004). It has been
1
This work was supported by the Scottish Crop Research
Institute (Marie Curie Training Ph.D. Fellowship in Plant Virology
no. QLK3–CT–2001–60032 to M.B.-P.). The Scottish Crop Research
Institute is supported by the Scottish Executive Environment and
Rural Affairs Department.
2
Present address: Department of Genetics and Plant Breeding,
Slovak University of Agriculture, 949 01 Nitra, Slovak Republic.
* Corresponding author; e-mail clacom@scri.sari.ac.uk; fax 44–
1382–562426.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.062810.
Plant Physiology, August 2005, Vol. 138, pp. 2155–2164, www.plantphysiol.org Ó 2005 American Society of Plant Biologists 2155
demonstrated that HSP90 interacts with RAR1 and
SGT1 and is essential for RPS2-mediated resistance in
Arabidopsis (Arabidopsis thaliana; Takahashi et al., 2003).
In addition, HSP90 also interacts with other R gene
products such as the resistance proteins N in Nicotiana
benthamiana and RPM1 in Arabidopsis (Hubert et al.,
2003; Liu et al., 2004). Many, but not all, of the powdery
mildew Mla R genes require the Rar1 gene for their
function (Freialdenhoven et al., 1994; Peterhansel et al.,
1997; Schulze-Lefert and Vogel, 2000), and Rar1 independence
is determined by a single-amino acid substitution
within the Leu-rich repeat of the barley MLA6
and MLA13 proteins (Halterman and Wise, 2004).
Sgt1 and Rar1 are required in multiple R genemediated
and nonhost resistance responses to a variety
of pathogens (Peart et al., 2002; Schornack et al., 2004).
So far, functional proof that Hsp90 is a required component
in disease resistance signaling pathways has
been demonstrated only for dicotyledonous plants,
using virus-induced gene silencing (VIGS) and Arabidopsis
mutants (Lu et al., 2003, 2004; Takahashi et al.,
2003). This highlights the need to develop novel
approaches allowing the functional characterization
of molecular mechanisms associated with host and
nonhost resistance mechanisms in cereals.
To date, functional studies of genes associated
with host and nonhost resistance in barley utilized
reverse-genetics approaches based on gain-of-function
(transient gene expression; Schweizer et al., 1999) or
loss-of-function using double-stranded RNA interference
(Schweizer et al., 2000), which is a form of posttranscriptional
gene silencing. These approaches are
restricted to biolistic delivery of gene or doublestranded
RNA constructs into single cells (for review,
see Panstruga, 2004). The single cell-restricted inhibition
of endogenous gene expression confirmed Mlo
as a suppressor of broad-spectrum Bgh resistance
(Schweizer et al., 2000), and the requirement of Rar1 and
Sgt1 as part of signaling cascades leading to race-specific
Bgh resistance (Azevedo et al., 2002). The resistancebreaking
phenotypes obtained using this assay comprise
successful Bgh penetration and haustorium formation,
elongating aerial hyphae, and, eventually, secondary hyphae
formation restricted to the close vicinity of the
silenced cell (Schweizer et al., 2000; Panstruga, 2004).
However, such a silencing approach relies on efficient
delivery, minimal damage to the bombarded epidermal
cells, and a relatively fast turnover of preexisting endogenous
protein (Panstruga, 2004).
Reverse-genetics approaches based on VIGS mediate
a homology-dependent degradation of the target RNA
within multiple cells and cell layers of systemic upper
uninoculated leaves and organs from the challenged
plants. Previous studies have indicated that the VIGS
systemic silencing response is concomitant with viral
replication and is closely associated with systemic
leaves supporting virus invasion and replication. This
was illustrated by using a potato virus X (PVX) VIGS
vector with a deleted 25-kD open reading frame
(PVX.D25K) resulting in phloem restriction of the virus
(Himber et al., 2003). When PVX.D25K was used as
a VIGS vector to silence endogenous mRNA, such
as phytoene desaturase (Pds) or Rubisco, whose downregulation
leads to a visible silencing phenotype, the
silencing response was confined to veins and 10 to 15
adjacent cells, indicating that the silencing response
exhibits short-range movement (Himber et al., 2003).
Barley stripe mosaic virus (BSMV) has recently been
developed as a VIGS vector for monocots (Holzberg
et al., 2002; Lacomme et al., 2003) and generates a robust
silencing response in barley. Previous studies have
highlighted the invasion pattern of a green fluorescent
protein (GFP)-tagged BSMV-based vector (Haupt et al.,
2001). It was observed that the virus escaped first from
major veins, and, following cell-to-cell spread through
the mesophyll tissues, the epidermal cells were then
infected (Haupt et al., 2001). The invasion of the
epidermis is an important characteristic of BSMV and
its further use as a reverse-genetic tool for the characterization
of genes associated with Bgh resistance, as
fungal structures are observed only on epidermal cells.
We report here the use of a BSMV-VIGS vector for
the characterization of genes known to be associated
with powdery mildew resistance in barley and show
a requirement for HSP90 in Rar1- and Sgt1-dependent,
Mla13-mediated resistance.
RESULTS
Identification of Barley Mla13 Cultivars Most Suitable
for BSMV-VIGS Functional Studies
The efficiency of BSMV-induced gene silencing is
dependent on the ability of the host plant to tolerate
virus accumulation. As the inheritance of resistance to
BSMV has been reported (Vasquez et al., 1974; Carroll
et al., 1979), it is necessary to identify appropriate
barley cultivars that provide a suitable genetic background
to study molecular mechanisms associated
with powdery mildew resistance (cultivars harboring
Mla R genes) and that tolerate the substantial levels of
BSMV accumulation required to elicit a significant
VIGS response. Barley cultivars Spire, Tyne, Clansman,
Pallas near-isogenic line P11, and Digger (Cereal
Pathogen Resistance Allele database http://www.
drastic.org.uk) harboring the Mla13 R gene were
screened to identify genotypes capable of supporting
BSMV replication and allowing functional characterization
of genes associated with host resistance to the
Bgh isolate 139 (National Institute of Agricultural
Botany [NIAB], Cambridge, UK), carrying the corresponding
avirulence gene AvrMla13.
We used a previously described BSMV.hpPDSHv60
construct (Fig. 1; Lacomme et al., 2003) designed to
silence Pds, a visual marker of gene silencing, to assess
Mla13 barley cultivars for their ability to develop a
BSMV VIGS response. All the above-mentioned cultivars
exhibited photobleaching symptoms when challenged
with BSMV.hpPDSHv60. However, symptoms
triggered by BSMV infection ranged from mild chloHein
et al.
2156 Plant Physiol. Vol. 138, 2005
rotic stripes to necrotic areas in upper leaves, and these
were also observed in plants challenged by BSMV.GFP
and BSMV.hpPDSHv60 constructs (data not shown).
Spire and Clansman displayed the strongest photobleaching
with mild BSMV infection symptoms in
systemic leaves (Fig. 2A, left and right sections), as
opposed to Digger, where weak photobleaching and
strong BSMV infection symptoms were observed (data
not shown).
To gain a better insight into the level of Pds silencing,
we estimated the efficacy of the silencing response
triggered by BSMV.hpPDSHv60 in these cultivars at the
molecular level. Semiquantitative reverse transcription
(RT)-PCR was performed to assess the relative Pds
mRNA levels using an endogenous gene (ubiquitin)
as an internal standard as described previously
(Lacomme et al., 2003).
RT-PCR tests were carried out on samples from
leaves with the most pronounced silencing phenotype.
Samples were taken from three plants infected with
each construct. Pds mRNA levels were assessed by
amplification of a portion of Pds upstream of the
sequences introduced into the viral vectors (‘‘Materials
and Methods’’; Lacomme et al., 2003). Lower levels of
Pds mRNA were detected from silenced tissues of
BSMV.hpPDSHv60-infected plants than from control
BSMV.GFP-infected tissues in all cases (Fig. 2B, top
section). For all samples, ubiquitin mRNA levels were
comparable (Fig. 2B, bottom section). The strongest
reduction of Pds RT-PCR product accumulation between
control and silenced samples was detected in
leaves from Spire, Tyne, and Clansman (Fig. 2B, cultivars
1, 2, and 3, top and bottom sections), in line with
the observation that these cultivars developed the
strongest phenotypic Pds VIGS response. As BSMV
infection symptoms were less obvious in Clansman
(Fig. 2A, plants challenged by BSMV.GFP), this cultivar
was chosen to investigate Bgh Mla13-mediated
resistance.
Systemic Silencing of Sgt1, Rar1, and Hsp90
To demonstrate the efficacy and robustness of the
BSMV VIGS system for functional characterization of
genes associated with Bgh resistance, we targeted
genes known to be involved in Mla-mediated resistance
pathways (Rar1 and Sgt1) and Hsp90, which is
associated with many Rar1- and Sgt1-dependent resistance
pathways in dicotyledonous plants, but for
which there is no such evidence in monocots (Azevedo
et al., 2002; Hubert et al., 2003; Takahashi et al., 2003).
We generated VIGS constructs with antisense cDNA
fragments from Sgt1, Rar1, and Hsp90 (BSMV.SGT1as,
BSMV.RAR1as, and BSMV.HSP90as; Fig. 1). First leaves
from the barley cultivar Clansman were then inoculated
(Fig. 3A) with each of these and control (BSMV.GFP)
constructs. Systemic leaves numbers 2 and 3 (Fig. 3A)
were harvested at different time points after BSMV
infection ranging from 7 d post inoculation (dpi) to
21 dpi. The silencing response of the targeted genes
was monitored at both the mRNA and protein levels.
By 7 dpi, reduced levels of Sgt1, Rar1, and Hsp90
mRNA were detected in systemic leaves from plants
Figure 1. Schematic representation (not drawn to scale) of the BSMV
genome organization and BSMV constructs engineered to express GFP
and Pds, Rar1, Sgt1, and Hsp90 cDNA fragments as inverted repeats or
in antisense orientation. Genomic organization of BSMV RNAs a, b,
and g were as described previously (Holzberg et al., 2002). RNA b is
a ba (coat protein) deletion mutation.
Figure 2. Initial screening of barley cultivars for BSMV VIGS response.
A, Phenotypic assessment of virus symptoms and silencing abilities of
BSMV. hpPDSHv60 in barley cultivars Spire (left) and Clansman (right) is
shown. Symptoms were recorded at 14 dpi with BSMV.GFP control and
BSMV.hpPDSHv60 VIGS constructs. B, RT-PCR-based assessment of
the silencing response obtained in barley cultivars. Semiquantitative
RT-PCR was carried out on cDNA originated from BSMV.GFP-infected
(C) and BSMV.hpPDSHv60-infected (S) barley cultivars Spire (1), Tyne
(2), Clansman (3), Pallas (4), and Digger (5). PCR products from the
silenced gene Pds (top) were run alongside ubiquitin (bottom) to ensure
equal amounts of template cDNA were used. Products were visualized
after 30 cycles and 40 cycles of PCR amplification for, respectively,
ubiquitin and Pds by gel electrophoresis alongside no template control
(NTC). The most silenced leaves and the corresponding leaves from,
respectively, BSMV.hpPDSHv60- and BSMV.GFP-infected plants from
three different plants were pooled.
VIGS Characterization of Barley Mildew Resistance Genes
Plant Physiol. Vol. 138, 2005 2157
challenged with corresponding BSMV VIGS construct,
compared to leaves from BSMV.GFP-infected control
plants (Fig. 3B, left section). No significant difference in
level of ubiquitin or Pds (data not shown), included as internal
control for the input of RNA, was observed (Fig. 3B,
left section). Reduced levels of Sgt1, Rar1, and Hsp90
mRNA were detected up to 21 dpi in systemic silenced
leaves, indicating that the silencing response was
stable for longer periods of time (Fig. 3B, right section).
Because protein stability may mask the silencing
effect observed at the mRNA level we therefore assessed
the accumulation of SGT1, RAR1, and HSP90
proteins in control and silenced leaves. Total protein
extracts sampled from control and silenced
Clansman leaves were analyzed by protein gel blots
using antisera to SGT1, RAR1, or HSP90. By 7 dpi
significantly lower levels of SGT1, RAR1, and HSP90
(approximately 45 kD, 28 kD, and 80 kD, respectively)
were detected in leaves of plants challenged with
the corresponding BSMV VIGS construct in comparison
to controls (Fig. 3C). These results indicate that the
VIGS response triggered by BSMV leads to a downregulation
of Sgt1, Rar1, and Hsp90 genes detectable at
both the RNA and protein levels.
We previously observed that the BSMV VIGS response
was also observed in emerging leaves originating
from axillary shoots by 21 dpi with BSMV.PDSas
(Holzberg et al., 2002; data not shown). We investigated
the efficacy of the VIGS response in these tissues
by monitoring HSP90, SGT1, and RAR1 protein levels.
By 21 d post BSMV inoculation, axillary leaves were
fully developed (up to three axillary leaves; Fig. 4A).
Protein extracts from silenced and control plants were
prepared using these axillary leaves. Reduced levels of
HSP90 (Fig. 4B) SGT1 (Fig. 4C), and RAR1 (Fig. 4D)
proteins were detected in axillary leaves challenged
with BSMV VIGS constructs, in comparison to control
axillary leaves. This indicates that axillary leaves still
display a significant silencing response and could provide
an alternative source of silenced tissues to characterize
molecular mechanisms associated with powdery
mildew resistance.
Resistance-Breaking Phenotypes of Powdery Mildew
on BSMV-Silenced Barley Leaves
As the different BSMV VIGS constructs were used to
assess the functional involvement of selected genes in
Mla13-mediated resistance to Bgh, the development of
fungal structures was observed and used as a criterion
to phenotypically assess resistance levels.
Leaves 2, 3, and 4 from silenced plants exhibiting mild
virus symptoms were detached at 7 dpi and 14 dpi
from the main stem, and leaves 1, 2, and 3 from axillary
Figure 3. Molecular assessment of BSMV-based VIGS efficacy on main stem leaves of barley cultivar Clansman. A, Schematic
representation of young barley plants 7 d post BSMV infection. The arrow points to the first emerging barley leaf on the main stem
that was used for virus infection. Systemic leaves 2 (L#2) and 3 (L#3) were harvested and prepared for RT-PCR and western-blot
analysis (not drawn to scale). B, RT-PCR results for Hsp90, Sgt1, and Rar1 following amplification with gene-specific primers
7 dpi (left) and 21 dpi (right) with BSMV constructs for silencing target genes (S) and in control plants infected with BSMV.GFP
(C). Equal template input is shown by PCR amplification with ubiquitin-specific primers. Systemic leaves (either L#2, or L#2 and
L#3) from at least two different plants were pooled. C, Western-blot analysis for HSP90, SGT1, and RAR1 on leaf number 2 at 7
dpi with BSMV.GFPas a control and BSMV VIGS vectors using the target gene-specific sequence (left). The position of molecular
mass standard Broad Range (for HSP90 and SGT1 western blotting; Bio-Rad) or DualVue Markers (for RAR1 western blotting;
Amersham) is indicated. Protein loading is shown by Ponceau staining (right). For both RT-PCR and western-blotting analyses,
pooled leaves from three different plants were sampled.
Hein et al.
2158 Plant Physiol. Vol. 138, 2005
shoots at 21 dpi (Figs. 3A and 4A) and inoculated with
Bgh. After 4 to 5 d post Bgh inoculation, leaves were
fixed and fungal structures stained (described in
‘‘Materials and Methods’’). Our observations focused
on the primary infection process of powdery mildew,
which consists of six morphologically identifiable
stages: spore germination, formation of appressorial
initials, maturation of appressoria, formation of penetration
pegs, formation of haustoria, and formation of
secondary hyphae. The formation of mature haustoria,
which is generally observed by 34 to 36 h post Bgh
inoculation and the development of secondary hyphae,
which form by 3 to 4 dpi, are prerequisites for
establishment of a compatible interaction between
host and parasite (Ellingboe, 1972). The percentage of
spores developing mycelial growth (hyphae) out of
100 observed spores per leaf was used as an indicator
of resistance levels. We compared the proportions of
spores developing hyphae on the genetically susceptible
barley cultivar Golden Promise (corresponding to
100% of relative penetration) to the genetically resistant
cultivar Clansman (not BSMV infected) and to
Clansman plants infected with various BSMV constructs.
The experiment was conducted three times for
main stem leaf tests, assessing hyphae growth on four
leaves of each type (main stem leaf numbers 2, 3, and
4) from four plants per construct. The experiment was
repeated twice for axillary leaves, sampling three
leaves of each type (axillary leaf numbers 1, 2, and 3)
and from three plants per construct.
Typical results are shown in Figure 5, and the levels
of susceptibility compared with the susceptible cultivar
Golden Promise are presented in Figure 6. On
main stems, hyphae growth was observed on all leaves
from the susceptible barley cultivar Golden Promise
lacking the cognate Mla13 R gene (Fig. 5A). On
average, 20 (61.5%) out of every 100 spores developed
hyphae in this compatible interaction and were defined
as 100% of relative penetration efficiency (Fig. 6).
Further data are relative to the 100% of relative penetration
in Golden Promise. In contrast, the majority of
Bgh spores developed only primary germ tubes and
appressoria prior to fungal growth arrest in the resistant
cultivar Clansman harboring the cognate Mla13
R gene (Fig. 5B). Indeed, only 10% (63%) of spores
inoculated on to leaves harvested from main stem
leaves could overcome the Mla13 resistance and establish
hyphae (Fig. 6, left section). Similarly, leaves
harvested from the barley cultivar Clansman inoculated
with BSMV.GFP and BSMV.PDSas prior to mildew
challenge revealed a Bgh resistance comparable to
that observed in non-BSMV-challenged plants, and
only 13% (65%) and 10% (62.5%) of spores developed
hyphal structures, respectively. Figure 5C shows a typical
fungal growth arrest in BSMV.GFP-infected plants
following Bgh inoculation. In contrast, Clansman
plants challenged with BSMV.SGT1as, BSMV.RAR1as,
and BSMV.HSP90as (Fig. 5, D–F) displayed a significant
increase in mycelial growth in all systemically
BSMV-infected tissues. The percentages of relative
penetration were 68% (69%), 36% (66.5%), and 40%
(65.5%) for plants challenged by BSMV.SGT1as,
BSMV.RAR1as, and BSMV.HSP90as, respectively
(Fig. 6, left section).
As the silencing response is observed in axillary
leaves by 21 dpi (Fig. 4), we also investigated the effect
of Sgt1, Rar1, and Hsp90 silencing in this tissue. As
observed for leaves of the main stem by 7 dpi and
14 dpi, control plants infected by BSMV.GFP or
BSMV.PDSas displayed no significant difference in
Figure 4. Molecular assessment of BSMV-VIGS efficacy on axillary leaves of barley cultivar Clansman. A, Schematic
representation of young barley plants 21 d post BSMV infection (not drawn to scale). The arrow points to the first leaf on the
main stem (corresponding to L1 from Fig. 3A) that was used for virus inoculation. B to D, Western-blot analysis for HSP90 (B),
SGT1 (C), and RAR1 (D) protein levels from axillary leaves at 21 dpi after BSMV.GFP, BSMV.HSP90as, BSMV.SGT1as, or
BSMV.RAR1as inoculation. Systemic leaves 1 (L1), 2 (L2), and 3 (L3; SGT1 and RAR1) or axillary leaf number 3 (HSP90) pooled
from three different plants was harvested and prepared for western-blot analysis. Protein loading is shown by Ponceau staining
(lower sections).
VIGS Characterization of Barley Mildew Resistance Genes
Plant Physiol. Vol. 138, 2005 2159
mycelial growth compared to non-BSMV-challenged
resistant Clansman plants. Specifically, 3% (63%), 3%
(61.5%), and 6% (66%) of relative penetration were
observed on unchallenged, BSMV.GFP-infected, and
BSMV.PDSas-infected Clansman, respectively (Fig. 6,
right section). In comparison to the main stem, significantly
lower percentages of relative penetration were
observed on the axillary leaves.
In silenced axillary leaves, the percentages of relative
penetration were 22% (62%), 23% (68%), and 46%
(624%) for BSMV.SGT1as-, BSMV.RAR1as-, and
BSMV.HSP90as-challenged plants, respectively. Our
results confirm that, in these tested foliar tissues,
BSMV-VIGS is an efficient approach to characterize
the functions of genes such as Sgt1 and Rar1 that are
required components of the Mla13-mediated resistance
response to Bgh. Furthermore, the resistance-breaking
phenotype observed in BSMV.HSP90as-silenced tissues
also implicates Hsp90 as a required component of Bgh
Mla-mediated resistance in barley.
DISCUSSION
In this study, we demonstrate that BSMV-VIGS is
a robust approach to identify genes associated with
Figure 6. Percentage of relative penetration following BSMV-mediated silencing of Sgt1, Rar1, and Hsp90 compared to control
plants. Note that 100% of relative penetration is defined as that observed in the susceptible barley cultivar Golden Promise.
Similarly, the number of spores developing hyphae for mock-inoculated cultivar Clansman (Resistant [Mla13]), for Clansman
infected with BSMV control constructs (BSMV.GFP and BSMV.PDSas), and for Clansman infected with BSMV VIGS constructs
(BSMV.SGT1as, BSMV.RAR1as, and BSMV.HSP90as) were reported as relative values with respect to Golden Promise controls.
Results presented are from three independent VIGS experiments using four different plants per construct and experiment (6SE),
corresponding to (A) main stem leaves (combined data from leaves 2 and 3 harvested at 7 d or leaves 2, 3, and 4 at 14 d post
BSMV challenge) and (B) axillary leaves (combined data from leaves 1, 2, and 3 harvested at 21 d post BSMV challenge).
Figure 5. Observation of fungal structures in silenced
and control leaves from main stem leaves 5 d post
Bgh inoculation. Fungal structures from Bgh isolate
139 carrying AvrMla13 were visualized by aniline
blue staining on leaves from (A) non-BSMV-infected
susceptible barley cultivar Golden Promise showing
highly developed hyphae, and (B) non-BSMVinfected
resistant barley cultivar Clansman (Mla13)
showing fungal growth arrest after primary germ tube
and appressorial germ tube development. Potential
effects of BSMV infection on Bgh resistance were
assessed by inoculating the barley cultivar Clansman
with the BSMV.GFP construct (C). Resistance breakdown
was observed on barley leaves of plants
infected with BSMV.SGT1as (D), BSMV.RAR1as (E),
and BSMV.HSP90as (F). The bars equal 200 mm.
Hein et al.
2160 Plant Physiol. Vol. 138, 2005
fungus resistance in barley and report that Hsp90 is
required for Bgh Mla-mediated resistance. We confirm
the requirement for Sgt1, Rar1, and Hsp90 genes in
the Mla13-mediated resistance response to powdery
mildew in barley, as previously observed in other R
gene-mediated responses in dicot species including
Arabidopsis and Nicotiana.
The involvement of Rar1 and Sgt1 in R-gene pathways
has been demonstrated in different plants
through either mutant analysis or transient loss-offunction
assays. The previously characterized barley
rar1 mutants are no longer resistant to Bgh in an Mla12
background (Freialdenhoven et al., 1994). For both
Rar1 and Sgt1, in planta functional studies in dicots
using VIGS (Liu et al., 2004) and in barley using singlecell
RNAi (Azevedo et al., 2002) have confirmed their
requirement in host resistance response. The BSMVVIGS
approach used in this study to silence Sgt1 and
Rar1 results in a resistance-breaking phenotype leading
to the development of secondary hyphae in
silenced leaves by 7 and 21 dpi with BSMV VIGS
constructs. The observed resistance-breaking phenotype
was comparable for both Rar1 and Hsp90, irrespective
of the development stage. Indeed, the
percentage of relative penetration was comparable in
silenced leaves from the main stem (by 7 dpi) and
axillaries (by 21 dpi). However, despite a clear role of
Sgt1 in Mla13-mediated resistance, a significant difference
in the percentage of relative penetration was
observed for Sgt1 silencing in the main stems (68% 6
6.5%) versus the axillary leaves (22% 6 2%). Further
experiments are required to conclude if a differential
tissue requirement for SGT1 could account for these
contrasting levels of resistance-breaking phenotype in
different types of leaves.
The BSMV VIGS approach has unequivocally established
the requirement of Hsp90 in Mla-mediated
resistance of barley to Bgh. The requirement of Hsp90
in host and nonhost resistance response was previously
reported using different approaches in model
plant systems. A high-throughput VIGS-based reversegenetic
screen in N. benthamiana has implicated
Hsp90 as a required component of N-, Rx-, and Ptomediated
resistance (Lu et al., 2003). Hsp90 was also
shown to be involved in a nonhost response in N.
benthamiana (Kanzaki et al., 2003). The physical interaction
of HSP90 with RAR1, SGT1, and the resistance
protein N has been demonstrated (Liu et al.,
2004). However, despite the fact that HSP90 interacts
with RAR1, SGT1, and the resistance protein RPM1 to
modulate its function, RAR1 and SGT1b can associate
independently with HSP90 (Hubert et al., 2003) in
Arabidopsis. Recently, it has been reported that the
stability of barley MLA resistance proteins (both
MLA1 and MLA6) is dependent on RAR1 (Bieri et al.,
2004). A similar RAR1 dependence was observed for
the stability of Rx protein in N. benthamiana providing
extreme resistance to PVX virus (Bieri et al., 2004).
Therefore, interactions among these proteins can regulate
R-protein function.
VIGS-based approaches allow the characterization
of genes such as Hsp90, for which down-regulation
leads in some cases to severe morphological defects, as
reported for N. benthamiana using tobacco rattle virus
(TRV) VIGS vectors (Liu et al., 2004) or PVX (Lu et al.,
2003), and therefore are likely to be lethal using RNAi
transgenic lines. No such severe phenotypes have been
observed in Hsp90-silenced barley. One could hypothesize
that either Hsp90 has different roles in
N. benthamiana and barley or that different levels of
Hsp90 down-regulation have been achieved, resulting
in different symptom severity. It is noteworthy that, in
the case of Sgt1 silencing, N. benthamiana Sgt1-silenced
plants using a TRV VIGS vector were shorter and more
branched than control plants (Peart et al., 2002). No
such obvious branching phenotype was observed in
barley despite significant reduction in SGT1 accumulation
in the main stem and axillary leaves. This could
simply be due to the architectural differences between
monocots and dicots or because Sgt1 is required for
pathogen resistance but not for plant development in
barley. Alternatively, this could be explained by the
ability of TRV to invade efficiently meristematic tissues
(Ratcliff et al., 2001; Valentine et al., 2004) and
therefore cause silencing at earlier stages of shoot
development than BSMV. However, BSMV strain
ND18, from which BSMV VIGS vectors were derived,
is seed transmissible, and other BSMV seed-transmitted
strains (such as MI-1) invade reproductive meristems
very early in their development (Johansen et al.,
1994). In our case, the BSMV VIGS vector used is
deleted from its coat protein (ba open reading frame).
Despite the fact that no data are available on the seed
transmissibility and meristem invasion of such mutants,
it has been reported that seed transmission
determinants are mapped on RNA g and RNA b
(Edwards, 1995). Considering the regulatory role of
the gb gene on RNA b expression (Petty et al., 1990), it
is conceivable that such a coat protein mutant may
display a reduced meristem invasion and prevent
severe developmental phenotypes associated with
Sgt1 silencing.
Another important factor that may influence functional
characterization and the silencing response is
the gene family complexity of Rar1, Sgt1, and Hsp90
in barley. In Arabidopsis, Sgt1 and Rar1 have been
identified as, respectively, two copies (Austin et al.,
2002; Azevedo et al., 2002) or a single-copy gene
(Muskett et al., 2002), whereas Hsp90 has seven copies
(four in the cytoplasmic subfamily; Krishna and Gloor,
2001). It is believed that Sgt1 and Rar1 are both singlecopy
genes in barley (Takahashi et al., 2003). Hsp90 is
likely to belong to a multiple-gene family in barley as
for Arabidopsis. Indeed, a BLASTN search (Altschul
et al., 1997) for barley expressed sequence tags (ESTs)
at the National Center for Biotechnology Information
database (http://www.ncbi.nlm.nih.gov/BLAST/)
with homology to the 383-bp cytosolic HvHsp90 sequence
used for the silencing experiments revealed
380 ESTs, from which 248 shared at least 25 nt
VIGS Characterization of Barley Mildew Resistance Genes
Plant Physiol. Vol. 138, 2005 2161
consecutive homology and therefore could be targeted
by the BSMV.HSP90as VIGS construct. Using Cap3
software (Huang and Madan, 1999), these ESTs could
be grouped into three different contigs, each homologous
to cytosolic HSP90. Therefore, at least three
barley Hsp90 cytosolic genes and another yet unidentified
barley cytosolic Hsp90 are likely to be
targeted by the BSMV.HSP90as construct. As significant
decreases in HSP90 protein level were observed,
we can conclude that the VIGS response generated
by the BSMV.HSP90as construct is likely to have
targeted several members of the barley cytosolic
Hsp90 family and may be robust enough to characterize
other genes present in multiple copy numbers.
BSMV fulfills several important characteristics for
use as a reverse-genetic tool for functional characterization
of genes associated with Blumeria resistance in
barley. We identified Mla cultivars that tolerate BSMV
accumulation and observed silencing without alteration
or masking of the host response to pathogen
challenge. We also demonstrated that BSMV challenge
does not affect Bgh resistance as the percentage of
relative penetration was similar in BSMV-uninoculated
and BSMV.GFP- or BSMV.PDSas-inoculated
plants. Consequently, BSMV infection is not likely to
interfere with molecular mechanisms associated with
secondary pathogen infection. In addition, the silencing
response was robust enough in the epidermis to
support Bgh colonization and was expressed in a sufficient
number of cells to allow mycelial growth.
Hyphae appear clustered on the leaf surface, possibly
because of the nonuniform nature of the silencing
response that could reflect the pattern of unloading
BSMV in systemic leaves. Previous studies have highlighted
the invasion pattern of a GFP-tagged BSMVbased
vector (Haupt et al., 2001). Inoculation of source
leaves of barley produced systemic symptoms on sink
leaves within 4 to 5 d following inoculation. BSMV
systemic movement mirrored the unloading pattern of
phloem-mobile fluorescent solute (carboxyfluorescein).
As in the case of carboxyfluorescein unloading,
the exit of BSMV.GFP was discontinuous along the
length of a single vein but covered the whole leaf. It
was observed that the virus escapes first from major
veins and then spreads cell to cell through the mesophyll
and the epidermis.
The systemic nature of the VIGS response allows the
rapid characterization of genes associated with local
and systemic resistance in barley and other cereals.
VIGS pervasiveness, in contrast to microprojectilemediated
RNAi approaches, which show phenotypes
for a limited amount of time after bombardment
(usually 4 d), allow characterization of genes whose
corresponding proteins have a relatively slow turnover.
In addition, all types of cells (both epidermal and
mesophyll cells) and newly developing leaves displayed
a significant systemic VIGS response. This
opens up the possibility of conducting further studies
of genes associated with a multilayered defense response
in local and systemic tissues.
MATERIALS AND METHODS
BSMV Constructs and Cloning of cDNAs
For VIGS experiments, the gRNA-based BSMV vector (Holzberg et al.,
2002; Lacomme et al., 2003) was utilized to silence Pds, Sgt1, Rar1, and Hsp90
genes in barley (Hordeum vulgare) as described previously (Lacomme et al.,
2003). VIGS constructs were engineered by cloning barley Sgt1, Rar1, and
Hsp90 fragments using gene-specific primers harboring NotI and PacI restriction
sites at their extremity. Primer sequences used to clone a 371-bp
portion of HvSgt1 (GenBank accession no. AF439974) were 5#-TTGCGGCCGCACATCAAGCTGGGCAGTTA-3#
as a forward primer and reverse primer
5#-TATTAATTAAGGCTTGCTTGGCACTTCTAC-3#. A 367-bp portion of HvRar1
(GenBank accession no. AF192261) was amplified by RT-PCR using the
forward primer 5#-GAGCGGCCGCAAGCAAAGAAGCCATGAT-3# and the
reverse primer 5#-GTTTAATTAATCCTTGTACGTTTTGCCACAT-3#. A fragment
of 395 bp in length for HvHsp90 (GenBank accession no. AY325266) was
amplified from plasmid pCC194 harboring the full-length barley cytosolic
HSP90 (HvHsp90) cDNA clone whose corresponding HvHSP90 protein interacts
with Sgt1 and Rar1 (Takahashi et al., 2003) using the forward primer 5#-AAAAGCGGCCGCAAGCAGAAGCCTATC-3#
and the reverse primer 5#-TATTTTAATTAATTCTTGACAAGGTTC-3#.
PCR-amplified cDNA fragments were
then digested with PacI and NotI and inserted into the g.bPDS4-as (Holzberg
et al., 2002) vector digested with PacI-NotI to generate constructs BSMV.SGT1as,
BSMV.RAR1as, and BSMV.HSP90as. For the screening of barley cultivars with
the strongest Pds VIGS response, the BSMV.GFP (Haupt et al., 2001), as
a control of infection, and BSMV.hpPDSHv60 (Lacomme et al., 2003) constructs
were used. The BSMV.GFP construct and BSMV.PDSas (Holzberg et al., 2002;
Lacomme et al., 2003) were used as control of BSMV infection for the Blumeria
experiments. Generation of BSMV infectious RNAs from cDNA clones and
inoculation procedures were as described previously (Holzberg et al., 2002).
RNA Extraction, cDNA Synthesis, and
Semiquantitative RT-PCR
Barley endogenous plant gene cDNA fragments were obtained from total
RNA extracted from frozen barley cultivar Clansman leaves using the Qiagen
RNeasy plant mini kit (Qiagen, Crawley, UK) as described previously
(Lacomme et al., 2003). DNaseI treatment (DNA-free kit; Ambion, Austin,
TX) and first-strand cDNA synthesis by oligo(dT) priming using SuperScript
II RNase H2
reverse transcriptase (Invitrogen Life Technologies, Paisley, UK)
were as described previously (Lacomme et al., 2003).
For semiquantitative RT-PCR analysis, primers that anneal outside the
region of the cDNA cloned into BSMV to trigger silencing were used to ensure
that only the endogenous mRNA was amplified. Ubiquitin cDNA was used as
an internal constitutively expressed control. First-strand cDNA was used as
a template for PCR amplification through 30, 35, 40, 45, and 50 cycles. As 30
cycles (ubiquitin, Hsp90), 40 cycles (Pds, Sgt1), or 50 cycles (Rar1) of amplification
corresponded to the log-linear phase of PCR product amplification in
the nonsilenced control samples, these conditions were selected for comparison
of relative accumulation of both target Pds, Sgt1, Rar1, Hsp90 and control
ubiquitin mRNAs in all samples. Primers for amplifying the 97-bp ubiquitin
PCR fragment (GenBank accession no. X04133) were 5#-GCAAGTAAGTGCCTGGTCATGA-3#
as forward primer and 5#-ACAACCAGACATGCTCCAACCT-3#
as reverse primer. Primers for amplifying the 113-bp Pds PCR
fragment (GenBank accession no. AY062039) were 5#-TGGAGCTTATCCCAATGTACAGAA-3#
as forward primer and 5#-TGTATTCCCCTGGCTTGTTTG-3#
as reverse primer. Primers for amplifying the 613-bp Sgt1 PCR
fragment (GenBank accession no. AF439974) were 5#-TCGGATCTGGAGAGCAAGGCCAAGGAGG-3#
as forward primer and 5#-ATCTGTTCACCAAAGTCAACAACCACGC-3#
as reverse primer. Primers for amplifying the 426-bp
Rar1 PCR fragment (GenBank accession no. AF192261) were 5#-TGATGGCATGAAAGAGTGGAGCTGTTG-3#
as forward primer and 5#-ATGCAGCATCATGGTTATCCTTCTCC-3#
as reverse primer. Primers for amplifying
the 664-bp Hsp90 PCR fragment (GenBank accession no. AY325266) were
5#-AATTTCTGACGATGAAGACGAGGAGGAGA-3# as forward primer
and 5#-AGAAGCTCAGCAATCTTGGTCCTGTTCTGG-3# as reverse primer.
Western-Blotting Experiments
Silenced and control leaves were ground in liquid nitrogen and mixed with
two volumes of grinding buffer supplemented with Complete Protease
Hein et al.
2162 Plant Physiol. Vol. 138, 2005
Inhibitor Cocktail (Roche Diagnostics, Lewes, UK) as described by Bieri et al.
(2004). Proteins were separated on 12% SDS-PAGE gels and blotted to
Immobilon-P membrane (Millipore, Bedford, MA) using the Mini-Protean II
system (Bio-Rad, Hercules, CA). Anti-SGT1 and anti-RAR1 antibodies have
been previously described (Azevedo et al., 2002). The HSP90 antibody is an
affinity-purified polyclonal antibody raised against a 17-amino acid peptide
mapping within amino acids 1 to 50 at the amino terminus of HSP90 from
Arabidopsis (Arabidopsis thaliana). This region is conserved between HSP90 in
dicots (such as Nicotiana benthamiana GenBank accession nos. AY368904 and
AY519499; Lycopersicon esculentum GenBank accession no. AAR12195) and
monocots (such as barley GenBank accession no. AY325266 targeted for
silencing; Triticum aestivum GenBank accession no. X98582; Oryza sativa
GenBank accession nos. AB111810 and AY077617; Zea mays GenBank accession
nos. S59780 and A48426). This antibody recognizes the N-terminal
conserved region of HSP90 (HSP90-a aE-17; Santa Cruz Biotechnology,
Heidelberg), as described previously (Lu et al., 2003; Liu et al., 2004). Blots
were incubated with primary antibodies against SGT1 (rat polyclonal,
1:2,000), RAR1 (rabbit polyclonal, 1:5,000), and HSP90a (goat polyclonal,
1:1,000; HSP90-a aE-17, Santa Cruz Biotechnology) followed by incubation
with respective secondary antibodies anti-rat, anti-rabbit, or anti-goat IgGperoxidase
conjugates (Amersham Biosciences, Little Chalfont, UK), and then
chemiluminescent autoradiography (ECL Plus; Amersham Biosciences).
Molecular weight standards Broad Range (Bio-Rad) or DualVue Markers
(Amersham, Uppsala) were run alongside.
Plant Growth
All work involving virus-infected material was conducted in containment
glasshouses under Scottish Executive Environment and Rural Affairs Department
license GM/243/2005.
Barley cultivar Golden Promise (susceptible to Bgh), and cultivars harboring
the Mla13 R gene, such as Clansman, Spire, Tyne (harboring Ml [La] and
Mla13), Pallas near-isogenic line P11, and Digger (harboring Mla13), were
grown, as described previously (Hein et al., 2004), in controlled environment
chambers at 22°C with a 16-h photoperiod in natural light supplemented with
sodium lights (light intensity ranging from 400–1,000 mmol m22
s21
).
Mildew Growth and Barley Leaf Inoculation
BSMV-infected and control barley leaves originating from the main stem
(leaves 2, 3, and 4) or from axillary shoots (leaves 1, 2, and 3) were harvested
after 7, 14, or 21 d post challenge with BSMV constructs. Leaf segments of
about 5 cm length were cut and immediately placed in boxes (to provide
containment, SEERAD GM/243/2005) containing 0.5% (w/v) distilled water
agar and 1 mM benzimidazole. The leaf segments were then inoculated with
an avirulent strain of powdery mildew (Bgh), 139 (Avr13; obtained from
NIAB), to give approximately 15 to 20 sporulating colonies per cm2
and
incubated at 15°C in continuous light (100 mmol m22
s21
).
Staining and Microscopy
Leaves were fixed, cleared, and stained to allow observation of both fungal
development and plant responses to attempted infection. A modified method
was used for this process (Carver et al., 1991). Leaves were fixed for 24 h on
filter paper soaked with 1:1 (v/v) ethanol:acetic acid and for 48 h on filter
paper soaked with lactoglycerol (1:1:1 [v/v] lactic acid:glycerol:H2O), and
stained with aniline blue 0.1% (w/v) in 0.1 M K3PO4. Excess dye was carefully
rinsed off with distilled water. Leaves were mounted onto microscope slides
with mounting media Histomount (VWR, Poole, UK) prior to microscopy.
Samples were observed under light microscope Nikon Optiphot I (Nikon,
Kingston, UK), and pictures were taken using a Color Video Camera KY-F55B
(Photonic Science, Robertsbridge, UK) using software ImageProPlus 4.1
(Media Cybernetics, Silver Spring, MD).
Availability of Biomaterials
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes,
subject to the requisite permission from any third-party owners of all parts of
the material. Obtaining any permission will be the responsibility of the
requester.
ACKNOWLEDGMENTS
We acknowledge Jane Shaw for technical assistance. We thank Amelia
Hubbard at NIAB for providing us with Bgh isolate 139. We thank our
colleagues Eleanor Gilroy, Peter Hedley, and Robbie Waugh for critical
reading of the manuscript.
Received March 14, 2005; revised May 6, 2005; accepted May 24, 2005;
published July 22, 2005.
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