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HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic

Fungal Pathogen Blumeria graminis W OA

Daniela Nowara,a Alexandra Gay,a,1 Christophe Lacomme,b,2 Jane Shaw,b Christopher Ridout,c Dimitar Douchkov,a
Götz Hensel,a Jochen Kumlehn,a and Patrick Schweizera,3
a Leibniz-Institute
of Plant Genetics and Crop Plant Research, 06466-Gatersleben, Germany
b ScottishCrop Research Institute, Invergowrie, DD2 5DA Dundee, Scotland
c John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom

Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of
plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and
virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the
accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting
fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-
induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development
in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the
silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to
silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an
RNAi-based crop protection strategy against fungal pathogens.

INTRODUCTION Plants control viral diseases by RNA interference (RNAi) and

Obligate biotrophic plant pathogens, such as powdery mildew
and rust fungi, represent a large group of organisms that together
cause diseases on thousands of plant species (Schulze-Lefert
and Vogel, 2000; Zhang et al., 2005). In sharp contrast with their
overall importance, little is known about genes and factors of
powdery mildew and rust fungi required for pathogenesis and
virulence (Zhang et al., 2005). Besides the lack of robust trans-
formation protocols, one of the obstacles to a better under-
standing of gene function in these pathogens is the fact that any
stable transformation event or mutation causing apathogenicity
or avirulence will be lost because it thus becomes impossible to
maintain them on living plant material. On the other hand, the
expected public release of the genome sequence of the barley
powdery mildew Blumeria graminis f. sp hordei (B. graminis) will
offer new opportunities for a better understanding of the molec-
ular basis of its host interaction provided postgenomic tools and
resources will also become available (http://www.blugen.org).
1 Current address: Saatzucht Steinach, Wittelsbacher Strasse 15,
D-94377 Steinach, Germany.
2 Current address: SASA, Roddinglaw Road, Edinburgh EH12 9FJ, UK.
3 Address correspondence to schweiz@ipk-gatersleben.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Patrick Schweizer
(schweiz@ipk-gatersleben.de).
W
Online version contains Web-only data.
OA
Open Access articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.110.077040
exhibit enhanced resistance when carrying suitable antisense
or hairpin RNAi constructs (Waterhouse and Fusaro, 2006;
Sudarshana et al., 2007). Recently, reduced development of root-
knot nematodes as well as Lepidoptera and Coleoptera insects
feeding on transgenic plants that carry RNAi constructs against
target genes in these pests has been reported (Huang et al., 2006;
Baum et al., 2007; Mao et al., 2007). The uptake of double-
stranded RNA (dsRNA) or small interfering RNA (siRNA) into these
animals occurs by sucking or chewing of plant material, followed
by resorption in the (mid)gut system. On the other hand, biotrophic
fungal pathogens such as B. graminis interact with their corre-
sponding host intimately via a highly specialized cell called a
haustorium, which is surrounded by the extrahaustorial matrix
bordered by plant and fungal membranes on either side and that
represents the interface for signal exchange as well as nutrient
uptake (Panstruga, 2003) (see Supplemental Figure 1 online). This
close contact of the interaction partners might facilitate not only
the exchange of proteins but also of RNA as a carrier of biological
information affecting the outcome of the interaction. This hypoth-
esis would have far-reaching implications for our understanding of
plant–pathogen interactions and disease control.
As in fungal or oomycete pathogens, parasitic plants also form
haustoria as feeding organs during their interaction with host
plants. Recently, exchange of mRNA and small noncoding RNAs
has been described between host and parasitic plants, such as
Cuscuta pentagona or Tryphisaria versicolor (Westwood et al.,
2009). These findings lend initial support to the hypothesis of
exchange of genetic information between plants and pathogens
via exchange of RNA molecules. Here, we tested the possibility
that silencing-inducing RNA molecules are exchanged between
cereal hosts and the obligate biotrophic fungal pathogen B.

The Plant Cell Preview, www.aspb.org ã 2010 American Society of Plant Biologists 1 of 12
2 of 12 The Plant Cell

Table 1. Summary of the Screening for Fungal Target mRNAs Affected

used RNAi in the host cell to target fungal transcripts and
by HIGS examined the progression of the fungal infection. We tested 76
fungal candidate genes that were found to be expressed in
Screening in Barley Genotype
planta during the interaction (Zierold et al., 2005). For this aim, we
RNAi Target mRNAs GP I mlo5
used a transient assay system based on biolistic bombardment
of RNAi constructs into single epidermal cells (Douchkov et al.,
All tested candidates 38 38 2005) (see Supplemental Figure 1 online). None of the RNAi
First-round candidates 13 (16) 14 constructs was predicted to possess off-targets in the barley
Confirmed candidates 10 6
transcriptome as determined by the SI-FI software tool (http://
RNAi constructs targeting candidate genes of B. graminis were cobom- labtools.ipk-gatersleben.de/). The 76 candidate mRNAs were
barded with pUbiGUS (for the detection of transformed cells by GUS targeted by host-induced gene silencing (HIGS) in B. graminis
staining) into barley epidermal cells, followed by inoculation with B.
growing either on cv Golden Promise (GP) or on cells of the
graminis 1 d (I mlo5) or 3 d (GP) later. RNAi constructs that reduced
resistant mutant Ingrid BC7 mlo5 (I mlo5) that were rendered
haustorium formation by at least 20% in I mlo5 or 33% in GP were
selected for a final number of at least five independent experiments. The
susceptible by transiently expressing the Mlo wild-type allele
number in parentheses in the GP screen includes three additional (Shirasu et al., 1999). The use of I mlo5 also allowed the
candidates selected randomly or due to sequence similarity to GTF quantification of hyphal growth rates without interference from
proteins. The RNAi effects of confirmed candidates differed significantly neighboring colonies growing out from nontransformed cells.
from the empty vector control (P < 0.05). The two screens, in which nonoverlapping sets of genes were
tested, revealed 16 out of 76 (21%) target mRNAs of B. graminis
that resulted in a significant reduction of the percentage of fungal
graminis by assessing fungal development on plants that ex- conidia able to form a haustorium upon introduction of the RNAi
press RNAi constructs directed against fungal target transcripts. construct into the host cell (Tables 1 and 2; see Supplemental
Table 1 online for full details). No RNAi construct was identified
that significantly altered the rate of secondary hyphae elongation
RESULTS on I mlo5 hosts (data not shown). The regulation of the gene
candidates, which had been selected for repeated experiments
Screening for Fungal RNAi Target Genes based on initial screening results, was analyzed using publicly
available transcript-profiling data from a B. graminis–barley
To determine whether the accumulation in barley (Hordeum interaction (Both et al., 2005a; see Supplemental Table 2 online).
vulgare) cells of dsRNA or siRNA targeting fungal transcripts As shown in Supplemental Figure 2 online, all candidates
could affect the outcome of the interaction with B. graminis, we represented on the array were transiently expressed during the

Table 2. Significant Disruption of Fungal Development by RNAi Constructs Targeting Fungal Genes

Clone IDa HIGS Screen Rel. HI (%)b Min.c nd Pe Short Description (BLASTX) E-Value

HO09I03 GP 82.3 6 3.3 75 5 0.0058 Hypothetical (Magnaporthe oryzae) 3 3 10 89

HO07E08 GP 76.0 6 8.0 38 11 0.0135 Hypothetical (N. crassa) 4 3 10 43

HO15J13 GP 74.9 6 8.7 20 20 0.0097 1,3-b-glucanosyltransferase 1 3 10 52

HO11N21 GP 72.1 6 7.6 47 7 0.0106 1,3-b-glucanosyltransferase 1 3 10 82

HO08J20 GP 71.3 6 8.5 50 5 0.0285 No match

HO04M11 GP 71.2 6 6.5 50 7 0.0043 Hypothetical (G. zeae) 5 3 10 18

HO15I23 GP 65.8 6 10.2 38 5 0.0286 ADP/ATP carrier protein 4 3 10 74

HO14N21 GP 64.4 6 8.0 38 8 0.0030 Heat shock protein 70 1 3 10 133

HO06C14 GP 59.6 6 5.9 36 11 <0.0001 NADH-ubiquinone oxidoreductase 1 3 10 111

HO06F11 GP 59.1 6 7.6 28 7 0.0017 Vacuolar Ser protease 2 3 10 55

HO03F17 I mlo5 82.5 6 4.4 70 5 0.0170 Endosomal cargo receptor 9 3 10 65

HO08A09 I mlo5 79.7 6 5.0 62 6 0.0096 Hypothetical (Magnaporthe oryzae) 1 3 10 22

HO10J21 I mlo5 76.3 6 6.6 63 5 0.0228 Hypothetical (Magnaporthe oryzae) 6 3 10 29

HO12C01 I mlo5 74.9 6 3.0 68 5 0.0011 40S Ribosomal protein 6 3 10 32

HO06N16 I mlo5 73.6 6 9.2 43 5 0.0458 No match

HO10G24 I mlo5 66.4 6 9.2 40 5 0.0215 No match
aClone ID from the CR-EST database (http://pgrc.ipk-gatersleben.de/cr-est/index.php).
bHaustorium index relative to the empty vector control set to 100%.
cMin, minimum value (in percent) of rel. HI observed among n independent experiments.
dNumber of independent experiments.
eP value for null hypothesis (no deviation from the empty vector control). The fungal origin of all RNAi target sequences was supported by an EST

sequence from in vitro–germinated B. graminis, except for HO06C14 that was instead supported by a highly similar sequence in the draft genomic
sequence of B. graminis.

interaction, with no bias for an expression peak of the confirmed
candidate genes early or late during the interaction. However,
mRNA levels of all confirmed candidates were detectable 4 to 15
h after inoculation, which is in agreement with the observed
reduction of haustorium formation occurring within the first 24 h
of the interaction.
Silencing of Fungal Glucanosyltransferase Genes
Two RNAi constructs reducing haustorium formation were de-
rived from the EST clones HO15J13 and HO11N21 (Kunne et al.,
2005) and target mRNAs with similarity to 1,3-b-glucanosyl-
transferase (GTF1 and GTF2). The occurrence of glycosylphos-
phatidylinositol-anchored GTF proteins appears to be restricted
to the fungal kingdom where they act in cell wall elongation and
as virulence factors (Caracuel et al., 2005; Mouyna et al., 2005).
In addition, GTF1 was found to belong to the penetration-
associated cap20 regulon of B. graminis (Both et al., 2005a)
(see Supplemental Figure 2 online). The encoded proteins fell
into two different phylogenetic clades of the fungal GTF gene
family that differ by the absence or presence of a Cys-rich
module near the C terminus of the protein (Ragni et al., 2007)
(Figure 1). We decided to address the function of GTF1 and GTF2
in a series of additional experiments including virus-induced
gene silencing (VIGS) of both genes in wheat and transgenic
barley plants that carry an RNAi construct against GTF1. Figure 2
shows that several constructs targeting nonoverlapping se-
quences of GTF1 or GTF2 significantly reduced haustorium
formation, which indicates target specificity of RNAi. These
results suggest a role of both GTF proteins in fungal host invasion
Figure 1. Unrooted Phylogenetic Tree Indicating Relationships of GTF1
and GTF2 Protein Sequences to GTF Reference Proteins from Different
Fungi Inferred by a Neighbor-Joining Analysis.
Bootstrap values (%) are indicated along branches. Common gene
names and accession numbers of GTF proteins are shown. The following
species abbreviations were used: Asfu, Aspergillus fumigatus; Caal,
Candida albicans; Fuox, Fusarium oxysporum; Magr, Magnaporthe
grisea; Pabr, Paracoccidioides brasiliensis; Sace, Saccharomyces cer-
evisiae.
Host-Induced RNAi in Powdery Mildew 3 of 12
Figure 2. HIGS of GTF1 and GTF2 Genes in B. graminis Affects Early
Fungal Development.
Reduction of haustorium formation induced by RNAi constructs that target
different regions of BgGTF mRNAs in the single-cell HIGS assay. Black
lines below the mRNA sequences show the length and locations of HIGS
target sequences, with exact start/end positions given above the line.
Rel. HI (%), haustorium index, relative to the empty vector control set to
100%. White, gray, and black boxes indicate poly(A) tails, noncoding
untranslated regions, and coding regions, respectively. Mean values of at
least five independent experiments in cv GP are shown, with P values for
significant difference from the empty vector control.
because a cross-silencing by either RNAi construct was not
predicted by the SI-FI tool.
VIGS mediated by the barley stripe mosaic virus (BSMV), a
tripartite sense-strand RNA virus resulting in dsRNA intermedi-
ates during its replication, has been established in barley and
wheat (Triticum aestivum; Holzberg et al., 2002; Scofield et al.,
2005) and was used here to further address the effect of silencing
of GTF-encoding genes during the interaction of wheat with the
wheat powdery mildew B. graminis f. sp tritici (B. graminis tritici).
This allowed addressing HIGS by an independent approach in a
related, second pathosystem of high agronomical importance.
Due to the irregular, patchy pattern of VIGS in wheat, randomly
chosen sites on third and younger leaves of BSMV-symptomatic
plants were selected for microscopy assessment of fungal
development, which resulted in mixed data from silenced and
nonsilenced sectors. B. graminis tritici inoculation of plants
infected with BSMV carrying a GTF1 antisense sequence of
774 bp resulted in a highly significant reduction of initial haus-
torium formation, whereas VIGS of GTF2 induced by an anti-
sense sequence of 468 bp significantly reduced the elongation
rate of secondary hyphae of B. graminis tritici (Figure 3). These
results suggest that the two GTF genes may be involved in
different steps of fungal development that could not be resolved
in the single-cell assay.
To test if HIGS can be used to protect transgenic plants from
fungal infection, we transformed barley with the hairpin RNAi
construct pIPKb007_BgGTF1 that targets GTF1 under the con-
trol of the maize (Zea mays) Ubiquitin-1 promoter (Himmelbach
et al., 2007). Several independent transgenic T1 populations
were tested for transgene expression and fungal development.
As shown in Figure 4, several lines carried both inverted repeats
of the RNAi construct. The observed weak and smeary hybrid-
ization signals of a GTF1-derived probe on RNA gel blots with
RNA from noninoculated plants indicate rapid degradation of the
4 of 12 The Plant Cell

Figure 3. VIGS of GTF1 and GTF2 Inhibits Fungal Development in the Wheat–B. graminis tritici Interaction.
Combined data from two to three independent experiments comprising 77 to 105 plants per construct are shown. ***, Significantly different from BSMV
wild-type control plants (a = 0.0001; Wilcoxon rank sum test of median); NS, not significant. Mean values 6 SE are also shown, although not used for
statistical analysis because data were not normally distributed. Elongating secondary hyphae were scored short (interaction type II) or long (interaction
type III) if their entire length was shorter or longer than 5 times the length of the conidiospore.

double-stranded transgene RNA. Three T1 lines exhibited sig-
nificantly reduced B. graminis disease symptoms, whereas
transgenic control line E26 that had lost the hairpin RNAi cassette
was as susceptible as wild-type control plants.
Silencing of Fungal Avra10 Gene
B. graminis appears to deliver the putative effector proteins
Avra10 and Avrk1 into host cells where they may support the
establishment of disease (Ridout et al., 2006). These proteins
are recognized by the matching resistance (R) gene products
MLa10 and MLk1 in some barley genotypes, which leads to
hypersensitive cell death stopping fungal invasion (Figure 5A).
Overexpression of Avra10 and Avrk1 in barley lacking the
corresponding resistance genes was found to increase fungal
invasion, suggesting indeed a role of the encoded proteins as
effectors (Ridout et al., 2006). We took advantage of the
predicted functions of Avrk1 and Avra10 inside host cells for a
series of proof-of-concept experiments of HIGS. As shown in
Table 3, HIGS of either effector gene caused a marked reduction
in haustorium formation in a susceptible host, thus supporting
the view that in the absence of corresponding R genes, these
proteins have effector function already early during the interac-
tion. On the other hand, transient overexpression of a synthetic
Avra10 gene induced cell death in the presence of Mla10 (Pallas
near-isogenic line P09), which was reflected by a reduced
number of epidermal cells accumulating anthocyanin induced by
the cobombarded reporter plasmid pBC17 (Schweizer et al.,
2000) (Figure 5B). The Mla10 gene induced a rapid hypersensi-
tive cell death reflected by a reduced formation of the first
haustorium (Figure 5C) and therefore was a suitable target for
assessing an R gene–dependent effect of silencing Avra10 via
the host. Figure 5D shows that HIGS of Avra10 reduced haustoria
formation in cv Pallas, whereas it had no effect in Pallas P09. The
unchanged haustoria formation in Pallas P09 probably reflects
the outcome of two opposite HIGS effects in this genotype that
neutralized each other: an increase in haustoria formation due to
escape from Mla10 recognition and a decrease due to the lack of
the Avra10 effector protein. It is important to note that, in the
presence of the Avra10 HIGS construct, the Mla10-dependent
effect on haustorium formation was eliminated (cf. the second
and fourth columns from the left). This is the predicted result
because in the absence of Avra10 (due to HIGS), the absence or
presence of the matching resistance gene in the host becomes
irrelevant for the interaction.
The reduction of target mRNA abundance has to be postulated
if RNAi is the underlying mechanism leading to a phenotypic
effect. However, to demonstrate this in the obligate biotrophic
fungus B. graminis is difficult because no reporter strains and
almost no information about gene function exist in this organism
due to its recalcitrance to genetic transformation. Therefore, no
known nondetrimental gene knockout phenotypes are available

Figure 4. Reduced Disease Symptoms by B. graminis on Transgenic
Barley Plants Carrying an RNAi Construct against GTF1.
T0 plants were analyzed by genomic PCR for the presence of the
selectable marker gene (Hptr) and of both inverted repeats (IR1 and IR2)
of the RNAi cassette. The expression of the hairpin RNAi construct in T1
lines was analyzed by RNA gel blotting. 26s rRNA, loading/blotting
control. Infection was estimated as percentage of leaf area covered by B.
graminis mycelium 7 d after inoculation according to Schweizer et al.
(1995) and is show here relative to nontransgenic control plants that were
set to 100%. Mean values 6 SE from five to six independent inoculation
experiments using a total of 116 to 170 T1 plants per line are shown. *,
Significantly different from control (a = 0.05).
for phenocopying by RNAi, which means that silencing of any
gene may compromise fungal development or pathogenicity.
This may result in a selection against efficiently silenced sporel-
ings during host interactions and, consequently, in difficulty to
observe target mRNA reduction in the surviving fungal cells. To
circumvent this problem, we analyzed Avra10 mRNA abundance
in fungal hausoria interacting with epidermal cells that expressed
both Avra10_RNAi and the matching Mla10 gene, which should
create a selection pressure for efficient silencing of Avra10 in
order to escape Mla10 detection. Furthermore, only fungal
haustoria interacting with transformed epidermal cells must be
analyzed, which was achieved by coexpressing wild-type Mlo in
the resistant barley line I mlo5 resulting in cell-autonomous
complementation of basal susceptibility (Shirasu et al., 1999).
Assuming that the Avra10 effector is also expressed in young
Host-Induced RNAi in Powdery Mildew 5 of 12
haustoria and not only at the prehaustorial stage, which has to be
postulated due to the HIGS effect on haustoria formation and
early (prehaustorial) recognition by the MLa10 protein (Figure 5),
RNA was extracted from bombarded and challenge-inoculated
leaves 24 h after inoculation following mechanical removal of
epiphytic fungal mycelium. This allowed for the selective extrac-
tion of fungal transcripts for TaqMan real-time RT-PCR analysis
from young haustoria, which are a likely target tissue of HIGS due
to their intimate interaction with host cells. Our assumption that
Avra10 is expressed in young haustoria is supported by an
expression time course that showed low expression in conidia
that just landed on the leaf surface, followed by rapid induction
within 6 h and high expression until 24 h after inoculation, with a
drop thereafter (see Supplemental Figure 3 online). Transient
expression of the fast-acting Mla10 allele resulted in a strong,
though not complete, reduction of the formation of the first
haustorium (Table 4), thereby creating a strong selection pres-
sure for Avra10 silencing that will counteract the selection
against silencing of this gene due to loss of effector functionality.
As a result, at least a partial reduction of Avra10 mRNA should
become visible assuming that the strength of HIGS is not equal
among a large population of haustoria but follows a normal
distribution. This assumption is supported by our observation
that with none of the tested fungal target genes a complete
elimination of fungal development occurred. Indeed, as shown in
Figure 6, the relative abundance of Avra10 transcript was signif-
icantly reduced by the Avra10 HIGS construct in the presence of
the cobombarded Mla10 overexpression construct strongly
suggesting target gene silencing inside the fungus. The unde-
tectable target transcript reduction in the absence of coex-
pressed Mla10 probably reflects inefficient Avra10 silencing in
those surviving haustoria that actually yielded the RNA for
transcript quantification. The nondetectable and partial RNAi
effect in the absence and presence of Mla10, respectively,
suggests indeed a virulence penalty resulting from Avra10 si-
lencing. At first sight, this is not in line with the frequent obser-
vation that effectors can be easily mutated or deleted by
pathogens without a significant loss of virulence. However,
Avra10 was found to belong to a complex gene family in B.
graminis with at least six clades (unisequences) differing by at
least 1% in DNA sequence, and the RNAi construct used here is
predicted by the SI-FI software to efficiently silence five out of
these six unisequences, which could explain the strong induced
phenotype (see Supplemental Table 3 online; Sacristan et al.,
2009). Taken together, our data provide direct evidence for HIGS
of the Avra10 effector family inside young B. graminis haustoria.
To further confirm target specificity of the observed effect of
Avra10 silencing, we performed an RNAi rescue experiment
(Figure 7) as described for the silencing of barley ubiquitin genes
(Dong et al., 2006). In this type of experiment, an RNAi construct
is coexpressed with a synthetic gene that encodes the target
protein but is insensitive to the RNAi effect due to a high density
of silent point mutations at codon wobble positions. The ex-
pected result of this experiment will be full complementation of
the RNAi phenotype by the RNAi rescue construct if the RNAi
phenotype is the result of specific silencing of one target mRNA
or of highly similar, functionally redundant target mRNAs. Here,
the RNAi construct pIPKTA30_Avra10 was cobombarded
6 of 12 The Plant Cell

Figure 5. The Phenotypic Effect of Avra10 Effector Silencing Depends on the R Gene Status of the Host.

(A) The B. graminis isolate used in this study (CH4.8) expresses both Avrk1 and Avra10 effector genes that are recognized by resistance genes Mlk1 and
Mla10 in Pallas BC lines P17 and P09, respectively. Effector recognition triggers a defense response, including cell death, which is seen as small dark
flecks in the P17 and P09 lines. Lack of Avr recognition produces a susceptible phenotype, seen in the Pallas line as white fungal pustules.
(B) Mla10-dependent cell death induced by transient expression of Avra10 in barley near isogenic line Pallas P09 (Mla10). cv Pallas (mla10) served as
negative control. Leaves were cobombarded with reporter plasmid pBC17 leading to anthocyanin accumulation 6 the Avra10 overexpression construct
pIPKTA9_Avra10, followed by counting of anthocyanin-stained cells 4 d after bombardment. Mean 6 SE from four biological replicates (two
independent experiments). Different letters inside or above columns indicate significant Avra10 effect (analysis of variance).
(C) Mla10 mediates a rapid resistance response in barley epidermis, resulting in a reduction of the formation of the first haustorium that is reflected by a
lower haustorial index (HI) of GUS-transformed epidermal cells. Mean 6 SE from five independent experiments is shown with P values for the
comparison of mean values (neighboring columns).
(D) HIGS of Avra10 eliminates the Mla10-mediated difference in initial haustorium formation by selectively reducing HI in Pallas lacking the R gene. Mean
values 6 SE from five independent experiments are shown with P values for the comparison of mean values (neighboring columns).

together with a synthetic Avra10 gene (pIPKTA9_Avra10_wob-
ble) that is saturated in silent point mutations by replacing B.
graminis with barley codons (Figure 8A). Figure 8B shows
that pIPKTA30_Avra10 caused a significant reduction of fun-
gal haustorium formation, whereas the rescue construct
pIPKTA9_Avra10_wobble alone was without effect. This sug-
gests that the level of Avra10 protein was not limiting during the
interaction between the used fungal isolate CH4.8 and barley
GP. Importantly, the cobombardment of pIPKTA30_Avra10
with pIPKTA9_Avra10_wobble eliminated the RNAi effect. The
Avra10 RNAi rescue construct had no effect on the efficiency
of cobombarded RNAi construct pPKTA36 that targets the
barley Mlo gene and phenocopies recessive mlo resistance
alleles (Schweizer et al., 2000). We conclude that the effects of
the Avra10_RNAi as well as the synthetic rescue constructs
were gene specific and demonstrate silencing of Avra10 in
B. graminis.
DISCUSSION
Based on the presented data, we postulate the transfer of dsRNA
or siRNA from host plant cells into powdery mildew fungi; these
RNAs can disturb the host–pathogen interaction by inducing
silencing of fungal housekeeping genes or genes required for
development or virulence. RNAs have been found to move
systemically in plants, including, for example, the movement of
viruses via plasmodesmata and phloem (Voinnet, 2005). How-
ever, no such structures have been observed across powdery
mildew haustorial walls (Hippe, 1985). Instead, intensive vesicle

Table 3. Reduction of Fungal Development by RNAi Constructs
Targeting Fungal Effector Genes
Construct Rel. HI (%)a Mina na Pb
pIPKTA30N 100 – 8
pIPKTA30N_Avrk1 53.6 6 10.0 18.0 8 0.0024
pIPKTA30N_Avra10 57.9 6 16.3 7.8 8 0.0368
aFor abbreviations, see Table 2.
bP value for null hypothesis (no deviation from the empty vector control).
fusion/budding at plant and fungal membranes of the haustorial
complex has been described, which is preceded by anterograde
and probably retrograde vesicle traffic in epidermal cells at sites
of fungal penetration (Hippe, 1985; An et al., 2006). In this
respect, exosomes are of special interest because they have
been described to accumulate in multivesicular bodies at plant-
powdery mildew contact sites and to be released into cell wall or
extrahaustorial matrix (Meyer et al., 2009). It is also important to
note that multivesicular bodies and exosomes from monocytes
and mast cell lines have been described to contain small non-
coding RNAs plus components of the silencing machinery, such
as GW182, and this is being discussed with respect to micro-
RNA-mediated intracellular gene silencing and as a mechanism
of cell-to-cell exchange of genetic information (Valadi et al.,
2007; Gibbings et al., 2009).
Theoretically, the observed phenotypes could also have been
caused by degradation in epidermal cells of mRNA that was
secreted from the fungus into the host. However, in the case of
seven target genes, including GTFI and II, silencing is likely to
have occurred inside B. graminis because the encoded proteins
clearly fulfill specific functions in fungal cells (Table 2). In the case
of Avra10, a role of the encoded signal-peptide free protein inside
host cells has been proposed, which could theoretically also be
explained by secretion of the corresponding fungal transcript
into the host (Shen et al., 2007). However, secretion of fungal
transcripts into host cells has not been reported so far, in
contrast with the secretion of signal-peptide free proteins
through a novel pathway independent from the conventional
endoplasmic reticulum–Golgi secretory pathway (Nickel and
Rabouille, 2009). In summary, our data are compatible with a
model of secreted dsRNA or siRNA trafficking from barley and
wheat into B. graminis probably via an exosomal pathway and
silencing of target genes inside the fungus.
The rate of ;20% of tested HIGS constructs that induced a
reduction in fungal haustorium formation appears relatively high
although the corresponding target genes had been preselected
for a likely role during host infection based on their expression
and, in many cases, upregulation in planta. One possible expla-
nation for the observed efficiency of the HIGS screening proce-
dure comes from the fact that plant multivesicular bodies/
exosomes are also observed at sites of primary germ tube
penetration, which occurs as early as 2 to 3 h after inoculation
(Hückelhoven et al., 2000). This means that the time interval for
the kicking-in of HIGS leading to a reduction of haustoria forma-
tion may be in the order of 12 to 18 h, which has been shown to be
sufficient for transient-induced gene silencing of the Mlo gene in
barley epidermal cells (Douchkov et al., 2005). Moreover, several
Host-Induced RNAi in Powdery Mildew 7 of 12
of the effective HIGS constructs target in planta–expressed B.
graminis genes with central housekeeping functions whose
silencing is predicted to be detrimental to the organism.
HIGS of the Avrk1 and Avra10 effector genes caused a
reduction in haustorium formation, which might be at odds with
the previously proposed release of such effectors by mature
haustoria (Panstruga and Dodds, 2009; Godfrey et al., 2010).
Blumeria species form not only the appressorial but also a
primary germ tube that has been shown to punch a hole into host
cell walls very rapidly after landing on its surface and suggested
to be important for water uptake and host recognition (Zhang
et al., 2005). Recently, a conditioning by the primary germ tube of
B. graminis of barley epidermal cells for enhanced susceptibility
to subsequent appressorial penetration and haustorium estab-
lishment in the same host cell has been reported (Yamaoka et al.,
2007). This indicates a very early release of effectors by the
primary germ tube and suggests a role of effector proteins not
only in maintenance of haustorium functionality and prevention
of hypersensitive death of invaded cells, but also in the inhibition
of very early defense responses, either at the prepenetration
phase or immediately after appressorial penetration pegs have
breached cell walls. Further support for this hypothesis comes
from the absence of Avra10 and Avrk1 transcripts from isolated
mature haustoria at 7 d after inoculation (Godfrey et al., 2010).
In summary, we propose HIGS as a novel tool to address gene
function in obligate biotrophic powdery mildew fungi that also
bears the potential of disease control by the use of transgenic
plants that express antifungal RNAi constructs. HIGS might be
used to control multiple diseases of a given crop because
constructs can be designed such as containing multiple stacked
RNAi target sequences. Alternatively, multiple transgenic events
can be stacked by crossing of corresponding lines. No side
effects of stacked events on host plants would be expected
provided careful selection of target RNAi sequences without off-
targets in the plant. We also propose to address in future
research the question of naturally occurring siRNA or miRNA
molecules trafficking between plants and fungal pathogens as
mediators of disease or resistance.
METHODS
Plants and Fungi
The barley (Hordeum vulgare) line I mlo5 was grown in pots containing soil
of the IPK nursery without fertilizers in the greenhouse at ;208C and a
photoperiod of 16 h by supplemental light from sodium halogen lamps.
Barley cv Golden Promise (GP), Pallas, and Pallas backcross lines P09
(Mla10) and P17 (Mlk1) were grown in pots containing soil of the IPK
Table 4. Resistance against B. graminis Isolate CH4.8 Mediated by
Transient Expression of Mla10
Constructs SIa n Pb
pUbiGUS + pU-Mlo 0.402 6 0.063 4 –
pUbiGUS + pU-Mlo + 0.112 6 0.015 4 0.0042
pENTR-(ubi)-Mla10-(c)
aSusceptibility index.
bP value for null hypothesis (no deviation from absence of Mla10).
8 of 12 The Plant Cell
Figure 6. Target Transcript Reduction by HIGS in B. graminis.
Transcript abundance of Avra10 in young fungal haustoria interacting
with transformed epidermal cells was quantified by TaqMan real-time
RT-PCR 24 h after bombardment with the Avra10 HIGS construct
(pIPKTA30_Avra10) in the absence or presence of coexpressed Mla10
[construct pENTR-(ubi)-Mla10-(c), here abbreviated as pMla10]. Basic
host compatibility of transformed cells of resistant line Ingrid BC mlo5
was established by coexpressing wild-type Mlo (construct pU-Mlo).
Twenty-four hours after B. graminis inoculation, poly(A)+ RNA was
extracted, reverse transcribed, and used for real-time PCR. Data repre-
sent mean values 6 SE from three independent quantitative PCR runs
using cDNA from two independent poly(A)+ preparations of one bom-
bardment experiment. Similar data were obtained from a second inde-
pendent biological replicate. The ratio of Avra10 transcript relative to the
monoglyceride lipase reference transcript is shown (Both et al., 2005b).
Statistical significance of the HIGS effect was determined by Student’s t
test (one-tailed; Avra10 HIGS construct versus empty vector control in
the absence or presence of Mla10 coexpression).
nursery without fertilizers in a growth chamber (16 h of light from metal
halogen lamps; 8 h of darkness; temperature 208C; relative humidity 60%
on average). The first leaves of 7-d-old GP or I mlo5 plants were used for
the particle bombardments. The pathogens Blumeria graminis f. sp hordei
(B. graminis, isolate CH4.8) and B. graminis f. sp tritici (B. graminis tritici,
isolate FAP93151) were cultivated on susceptible barley and wheat
(Triticum aestivum) plants, respectively, in separate climate chambers at
228C, 70% relative humidity, and a photoperiod of 16 h. Six to seven days
after inoculation, B. graminis or B. graminis tritici conidiospores were
used for the inoculation of detached leaves or experimental plants by
shaking diseased plants in an inoculation tower.
HIGS Single-Cell Assay
The RNAi constructs were designed using pIPKTA38 as entry vector and
pIPKTA30N as Gateway destination vector, and the single-cell assay
based on microprojectile bombardment was performed using first leaves
of 7-d-old barley plants, as described (Douchkov et al., 2005) (see
Supplemental Table 4 and Supplemental Figure 4 online). Approximately
400 to 500 bp of each inverted repeat sequence was contained by the
final constructs. Barley leaf segments of GP and I mlo5 were inoculated
with B. graminis 3 and 1 d after bombardment, respectively. Forty hours
after inoculation, the leaves were stained for the b-glucuronidase (GUS)
reporter gene and used for microscopy assessment of haustorium
formation (see Supplemental Figure 1 online). The haustorial index of
transformed cells expressing the cobombarded GUS reporter gene was
calculated by dividing the number of counted haustoria by the number of
blue-stained cells per bombardment.
RNAi Rescue
The RNAi rescue construct pIPKTA9_Avra10_wobble was made by
inserting a synthetic gene of 885 bp (Eurofins MWG Operon) into the
XbaI and XhoI sites of the transient expression vector pIPKTA9. The
synthetic Avra10 cDNA was designed such as to be mutated on the third
wobble position of each redundant codon by replacing the original B.
graminis codon usage by the most or second most common barley
codon. In silico testing by Si-Fi software of the synthetic gene for silencing
by the Avra10 HIGS construct pIPKTA30_Avra10 did not reveal any
21mer oligonucleotide as potential target. Therefore, we assume this
rescue construct to be fully immune against Avra10 silencing.
VIGS
cDNA Clones in pBluescript of a coat protein–deleted mutant of the
BSMV isolate ND18 were used for the construction of recombinant
versions of the g-genome containing antisense sequences of target
genes and for in vitro transcription of capped viral RNA, as described
(Bruun-Rasmussen et al., 2007). Inserts for constructs pBSMV_BgGTF1
(g-T7) and pBSMV_BgGTF2(g-T7) were PCR amplified using B. graminis
tritici DNA and the EST-clone HO09P14 as template, respectively. See
Supplemental Table 4 online for primer sequences. The fragments were
introduced into the BamHI and XmaI sites of pBSMV_MCS(g-T7)ND18
(Bruun-Rasmussen et al., 2007) in antisense orientation. Five-day-old
wheat plants (cv Kanzler) were used for virus inoculation by rubbing the
first leaf with 20 mL of 5% bentonite solution until dark green stripes
appeared on the leaf. Six microliters of an equimolar mixture of the three
different transcripts of the BSMV genome were then rubbed onto the
wounded leaf, followed by spraying briefly with water and incubation in a
growth chamber (16 h light at 258C and 8 h darkness at 208C, 60% relative
humidity). Seventeen to nineteen days after BSMV inoculation, all leaves
expect the first and second were harvested from plants showing BSMV
symptoms. Leaf segments (;6 cm long) were placed onto 1% phytoagar
(Duchefa) containing 0.002% benzimidazol. The leaf segments were
inoculated with B. graminis tritici at a density of 10 to 15 conidia mm22
and incubated for 40 h at 208C in a climatized room with indirect daylight.
Epiphytic fungal structures were stained with Coomassie Blue R 250 as
described (Seiffert and Schweizer, 2005).
Transgenic Plants
The binary RNAi construct pIPKb007_BgGTF1 was generated using the
RNAi vector pIPKb007 (Himmelbach et al., 2007; see Supplemental Figure
4 online). The LR reaction with entry clone pIPKTA38_BgGTF1 that
contained 833 bp of BgGTF1 cDNA sequence was performed as described
(Douchkov et al., 2005). Immature barley embryos (GP) were transformed
with the binary RNAi vector described above using the Agrobacterium
tumefaciens strain AGL1 as described (Hensel et al., 2008). The resulting
plantlets were selected on medium containing hygromycin (50 mg L21).
Approximately 15 individual T1 plants per selected primary transgenic line
were used for one inoculation experiment in 54-well multipot trays, together
with GP wild-type plants. Transgenic lines were arranged in rows, with
regularly interspersed wild-type control plants. Sixteen-day-old seedlings

Figure 7. Model of HIGS and RNAi Rescue of Avra10 in the Barley–B. graminis Interaction.
(A) Overview of interaction-related cellular structures ;16 h after inoculation. Please note that cell wall penetration by the primary germ tube and targeted
host secretion leading to a cell wall apposition are occurring ;10 h prior to appressorial penetration. Bgh, B. graminis; MVB, multivesicular bodies.
(B) to (D) Model of silencing and RNAi rescue of Avra10.
were inoculated with B. graminis by blowing conidiospores from four
infected leaves into an inoculation tower. Disease was scored 7 d after
inoculation, as described (Schweizer et al., 1995).
Fungal Transcript Analysis
Primary leaves of barley Ingrid BC mlo5 were cobombarded with a
mixture of plasmids containing pU-Mlo (Shirasu et al., 1999), empty RNAi
vector pIPKTA30N, or the HIGS construct pIPKTA30_Avra10, plus op-
tionally pENTR-(ubi)-Mla10-(c) (Seeholzer et al., 2010). Twenty-four hours
after bombardment, leaves were inoculated with B. graminis, and epi-
phytic fungal mycelium was wiped off by wet cotton pads prior to RNA
extraction 24 h after inoculation. The RNA from leaves and fungal
haustoria inside transformed epidermal cells with restored Mlo suscep-
tibility was isolated with the RNeasy kit (Qiagen). Poly(A)+ RNA was
isolated using magnetic Dynabeads (Invitrogen Dynal), DNase treated by
DNA-free kit (Ambion), and reverse-transcribed by oligo(dT) and random
priming into cDNA using the iScript cDNA synthesis kit (Bio-Rad).
Quantitative real-time PCR was performed in a volume of 15 mL using
Maxima Probe qPCR Mastermix (Fermentas) and an ABI 7900HT fast
real-time PCR system (Applied Biosystems). Forty cycles (15 s 3 948C, 30
s 3 568C, 30 s 3 728C, preceded by standard denaturation steps) were
conducted. Data were analyzed by the DCt method using the SDS 2.2.1
software (Applied Biosystems). For this purpose, standard curves were
Host-Induced RNAi in Powdery Mildew 9 of 12
included for each gene, with fivefold dilutions and three technical repli-
cates per cDNA sample. The reverse PCR primer and TaqMan probe of
Avra10 was placed outside the inverted repeat region of HIGS construct
pIPKTA30_Avra10 to prevent amplification of dsRNA. This was confirmed
by the absence of amplification products from RNA of leaf segments
bombarded with the Avra10 HIGS construct without subsequent B.
graminis inoculation (data not shown). For PCR primers and TaqMan
probe sequences, see Supplemental Table 4 online.
Phylogenetic Analysis of GTF Proteins
Amino acid sequences of GTF1 and 2 together with other GTF proteins
were aligned with ClustalX (Larkin et al., 2007) using the Slow-Accurate
algorithm with the BLOSUM series protein weight matrix and a gap
opening penalty of 10 together with a gap extension penalty of 0.1.
Afterwards, the alignment was checked and manually adjusted. Phylo-
genetic analyses using (1) neighbor-joining clustering based on pairwise
mean character differences and (2) maximum parsimony with the heu-
ristic search algorithm and TBR branch swapping were conducted in
PAUP* 4b10 (Swofford, 1993). Statistical support of branches was
assessed by bootstrap analyses using 1000 resamples of the data matrix.
As the outcomes of both analyses were nearly identical, only the unrooted
neighbor-joining tree is shown. The protein sequence alignment used for
tree construction is shown in Supplemental Data Set 1 online.
10 of 12 The Plant Cell

Figure 8. Rescue from Silencing of Avra10 by a Synthetic Gene Restores Fungal Haustorium Formation.
(A) Alignment of Avra10 wild-type (top) and synthetic Avra10_wobble (bottom) DNA sequences. Mismatches are highlighted by white boxes.
(B) Barley leaf segments were cobombarded with plasmid combinations followed by inoculation with B. graminis, and haustorium formation was
assessed microscopically. Rel. HI, haustorial index relative to the empty vector control (cobombardment of pIPKTA9 and pIPKTA30) set to 100%. Mean
values 6 SE from eight independent experiments are shown with P values for the null hypothesis. Construct pIPKTA340_Avra10 was used for Avra10
silencing in B. graminis, construct pIPKTA30_Mlo for silencing of barley Mlo was used as positive RNAi control, and construct pIPKTA9_Avra10_wobble
was used for RNAi rescue. For a schematic representation of constructs, see Supplemental Figure 4 online.

Statistical Analysis of HIGS Effects
Because the absolute susceptibility levels of empty vector or wild-type
controls varied between independent experiments, effects of RNAi or
antisense constructs were normalized to these internal controls (set to
100%) in each experiment. Deviations from hypothetical value 100 were
tested by one-sample t test or Wilcoxon rank sum test, depending on
normal distribution of data. All tests were performed two-sided. Unless
otherwise specified, a was set to 0.05.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: GTF1 of B. graminis f. sp hordei, EU646133; GTF1 of B.
graminis f. sp tritici, FJ422119; GTF2 of B. graminis f. sp hordei,
HQ234876; and synthetic gene Avra10_wobble, FJ422120.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Examples of Interaction Types in the HIGS
Single-Cell Assay of Barley and in the VIGS Assay of Wheat.
Supplemental Figure 2. Regulation in Planta of Tested HIGS Target
Genes of B. graminis.
Supplemental Figure 3. Time Course of Avra10 Transcript Levels in
B. graminis.
Supplemental Figure 4. Constructs Used for HIGS in the Single-Cell
Assay, by VIGS, and in Stable Transgenic Plants.
Supplemental Table 1. Results from the Initial HIGS-Based Screen-
ing of All 76 Tested Candidate Genes and from Repeated Experi-
ments of Selected Candidates.
Supplemental Table 2. BLASTN-Based Linking of cDNA Clones
Used in This Study to Clones Spotted onto a B. graminis cDNA Array
(13), and Log2-Transformed Average Signal Intensities of Selected
Transcripts.
Supplemental Table 3. Prediction of Avra10-Like Off-Target Tran-
scripts in B. graminis
Supplemental Table 4. PCR Primers Used for Construct Generation
and RT-PCR.
Supplemental Data Set 1. Fasta File of Alignment Used to Generate
the Phylogenetic Tree in Figure 1.
ACKNOWLEDGMENTS
The technical assistance of Sonja Gentz, Stefanie Lück, and Cornelia
Marthe (Leibniz-Institute of Plant Genetics and Crop Plant Research

[IPK]) is acknowledged as well as support of the VIGS work by Ingo Hein
(Scottish Crop Research Institute). We thank Merete Albrechtsen
(Aarhus University, Denmark) for supplying BSMV vectors, Tina Jordan
(Zurich University) for the kind gift of the Mla10 expression plasmid,
Frank Blattner IPK for calculating phylogenetic relationship of GTF
proteins, and I. Schubert IPK for improving the manuscript. This work
was supported by the Leibniz-Institute of Plant Genetics and Crop Plant
Research (to P.S.) and by EU-FP6 BIOEXPLOIT (to C.R.).
Received June 2, 2010; revised August 11, 2010; accepted September
14, 2010; published September 30, 2010.
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 HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria
graminis
Daniela Nowara, Alexandra Gay, Christophe Lacomme, Jane Shaw, Christopher Ridout, Dimitar
Douchkov, Götz Hensel, Jochen Kumlehn and Patrick Schweizer
PLANT CELL published online Sep 30, 2010;
DOI: 10.1105/tpc.110.077040

This information is current as of November 9, 2010

Supplemental Data http://www.plantcell.org/cgi/content/full/tpc.110.077040/DC1

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ADVANCING THE SCIENCE OF PLANT BIOLOGY