HIGS: Host-Induced Gene Silencing in the Obligate BiotrophicFungal Pathogen Blumeria graminis W OA

DanielaNowara,aAlexandraGay,a,1ChristopheLacomme,b,2 JaneShaw,bChristopherRidout,cDimitarDouchkov,a

Gotz Hensel,a Jochen Kumlehn,a and Patrick Schweizera,3

a Leibniz-Institute of Plant Genetics and Crop Plant Research, 06466-Gatersleben, Germanyb Scottish Crop Research Institute, Invergowrie, DD2 5DA Dundee, Scotlandc 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

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).

Plants control viral diseases by RNA interference (RNAi) and

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.

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 thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Patrick Schweizer(schweiz@ipk-gatersleben.de).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.110.077040

The Plant Cell, Vol. 22: 3130–3141, September 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

graminis by assessing fungal development on plants that ex-

press RNAi constructs directed against fungal target transcripts.

RESULTS

Screening for Fungal RNAi Target Genes

To determine whether the accumulation in barley (Hordeum

vulgare) cells of dsRNA or siRNA targeting fungal transcripts

could affect the outcome of the interaction with B. graminis, we

used RNAi in the host cell to target fungal transcripts and

examined the progression of the fungal infection. We tested 76

fungal candidate genes that were found to be expressed in

planta during the interaction (Zierold et al., 2005). For this aim, we

used a transient assay system based on biolistic bombardment

of RNAi constructs into single epidermal cells (Douchkov et al.,

2005) (see Supplemental Figure 1 online). None of the RNAi

constructs was predicted to possess off-targets in the barley

transcriptome as determined by the SI-FI software tool (http://

labtools.ipk-gatersleben.de/). The 76 candidate mRNAs were

targeted by host-induced gene silencing (HIGS) in B. graminis

growing either on cv Golden Promise (GP) or on cells of the

resistant mutant Ingrid BC7 mlo5 (I mlo5) that were rendered

susceptible by transiently expressing the Mlo wild-type allele

(Shirasu et al., 1999). The use of I mlo5 also allowed the

quantification of hyphal growth rates without interference from

neighboring colonies growing out from nontransformed cells.

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

conidia able to form a haustorium upon introduction of the RNAi

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

on I mlo5 hosts (data not shown). The regulation of the gene

candidates, which had been selected for repeated experiments

based on initial screening results, was analyzed using publicly

available transcript-profiling data from a B. graminis–barley

interaction (Both et al., 2005a; see Supplemental Table 2 online).

As shown in Supplemental Figure 2 online, all candidates

represented on the array were transiently expressed during the

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

by HIGS

RNAi Target mRNAs

Screening in Barley Genotype

GP I mlo5

All tested candidates 38 38

First-round candidates 13 (16) 14

Confirmed candidates 10 6

RNAi constructs targeting candidate genes of B. graminis were cobom-

barded with pUbiGUS (for the detection of transformed cells by GUS

staining) into barley epidermal cells, followed by inoculation with B.

graminis 1 d (I mlo5) or 3 d (GP) later. RNAi constructs that reduced

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

number in parentheses in the GP screen includes three additional

candidates selected randomly or due to sequence similarity to GTF

proteins. The RNAi effects of confirmed candidates differed significantly

from the empty vector control (P < 0.05).

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.

Host-Induced RNAi in Powdery Mildew 3131

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 ofGTF1 andGTF2

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 againstGTF1. 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 bothGTF proteins in fungal host invasion

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) andwas 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

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.

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.

3132 The Plant Cell

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 ofMla10 (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 ofAvra10 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 fromMla10 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 targetmRNA 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 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.

Host-Induced RNAi in Powdery Mildew 3133

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 Avra10mRNA 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-typeMlo 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

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

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).

3134 The Plant Cell

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

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 genesMlk1 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 accumulation6 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)Mla10mediates 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 theMla10-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).

Host-Induced RNAi in Powdery Mildew 3135

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

ofAvra10, 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

(Huckelhoven et al., 2000). This means that the time interval for

the kicking-in of HIGS leading to a reduction of haustoria forma-

tionmay be in the order of 12 to 18 h,which has been shown to be

sufficient for transient-induced gene silencing of theMlo gene in

barley epidermal cells (Douchkov et al., 2005). Moreover, several

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 mlo5was 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 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).

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 +

pENTR-(ubi)-Mla10-(c)

0.112 6 0.015 4 0.0042

aSusceptibility index.bP value for null hypothesis (no deviation from absence of Mla10).

3136 The Plant Cell

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 pathogensBlumeria 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 258Cand 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 roomwith 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 bpofBgGTF1 cDNAsequencewasperformed 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 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).

Host-Induced RNAi in Powdery Mildew 3137

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-timePCR system (Applied Biosystems). Forty cycles (15 s3 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

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 datamatrix.

As the outcomes of both analyseswere 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.

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.

3138 The Plant Cell

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 Luck, and Cornelia

Marthe (Leibniz-Institute of Plant Genetics and Crop Plant Research

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

assessedmicroscopically. 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 barleyMlowas 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.

Host-Induced RNAi in Powdery Mildew 3139

[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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Host-Induced RNAi in Powdery Mildew 3141

DOI 10.1105/tpc.110.077040; originally published online September 30, 2010; 2010;22;3130-3141Plant Cell

Douchkov, Götz Hensel, Jochen Kumlehn and Patrick SchweizerDaniela Nowara, Alexandra Gay, Christophe Lacomme, Jane Shaw, Christopher Ridout, Dimitar

graminisBlumeriaHIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen

 This information is current as of December 16, 2020

 

Supplemental Data /content/suppl/2010/09/14/tpc.110.077040.DC1.html

References /content/22/9/3130.full.html#ref-list-1

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