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Link to original content: https://pubmed.ncbi.nlm.nih.gov/20884801
HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis - PubMed Skip to main page content
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. 2010 Sep;22(9):3130-41.
doi: 10.1105/tpc.110.077040. Epub 2010 Sep 30.

HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis

Daniela Nowara  1 , Alexandra Gay, Christophe Lacomme, Jane Shaw, Christopher Ridout, Dimitar Douchkov, Götz Hensel, Jochen Kumlehn, Patrick Schweizer
Affiliations

HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis

Daniela Nowara et al. Plant Cell. 2010 Sep.

Abstract

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.

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Figures

Figure 1.
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 cerevisiae.
Figure 2.
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.
Figure 3.
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 (α = 0.0001; Wilcoxon rank sum test of median); NS, not significant. Mean values ± 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.
Figure 4.
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 ± 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 (α = 0.05).
Figure 5.
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 ± the Avra10 overexpression construct pIPKTA9_Avra10, followed by counting of anthocyanin-stained cells 4 d after bombardment. Mean ± 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 ± 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 ± se from five independent experiments are shown with P values for the comparison of mean values (neighboring columns).
Figure 6.
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 represent mean values ± se from three independent quantitative PCR runs using cDNA from two independent poly(A)+ preparations of one bombardment experiment. Similar data were obtained from a second independent 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).
Figure 7.
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.
Figure 8.
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 ± 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.
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