(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A,B) Defense-related salicylate- and jasmonate-responsive genes are not induced by <i>CYP3</i>-dsRNA.

<p>Detached second leaves of three-week-old barley were sprayed with 20 ng μL^-1 <i>CYP3</i>-dsRNA or TE (control), respectively, and 48 h later drop-inoculated with Fg-IFA65. Leaves were harvested 6 dpi and analyzed for gene expression by qPCR: <b>(A)</b> <i>Pathogenesis-related</i> 1 (<i>HvPR1</i>) and <b>(B)</b> <i>S-adenosyl-l-methionine</i>:<i>jasmonic acid carboxyl methyltransferase</i> (<i>HvJMT</i>). Both genes are highly responsive to Fg-IFA65 but not to <i>CYP3</i>-dsRNA or TE treatment. Please note that a combined treatment of <i>CYP3</i>-dsRNA followed by Fg-IFA65 48 h later also did not induce these marker genes, which shows independently that fungal development on <i>CYP3</i>-dsRNA-treated leaves is strongly inhibited.</p

(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A,B) Northern gel blot analysis of <i>CYP3</i>-dsRNA and <i>CYP3</i>-dsRNA-derived siRNA accumulation in local and distal (semi-systemic) barley leaf areas.

<p><b>(A)</b> Detection of 791 nt long <i>CYP3</i>-dsRNA precursor in pooled leaf tissue from non-infected leaves using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. Local (L) and distal (semi-systemic [S]) leaf segments were sampled separately at the indicated times after spraying with <i>CYP3-</i>dsRNA. No signal was detected in samples from TE-sprayed plants. <b>(B)</b> Recording <i>CYP3</i>-dsRNA-derived small RNAs in local and distal (semi-systemic) leaf areas using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. In this experiment, small RNAs could not be detected in distal (non-sprayed) tissues. siRNA generated <i>in vitro</i> by a commercial Dicer preparation from <i>CYP3</i>-dsRNA was used as positive control. No signal was detected in samples from TE-sprayed plants. Ethidium bromide-stained rRNA served as the loading control. Signals originate from the same membrane but different exposure times.</p

(A-D) SIGS-mediated semi-systemic control of <i>Fusarium graminearum</i>.

<p><b>(A)</b> Upper parts of detached second leaves of three-week-old barley were sprayed evenly with <i>CYP3</i>-dsRNA, TE, and <i>GFP</i>-dsRNA, respectively. After 48 h, the non-inoculated, semi-systemic (distal) tissue was drop-inoculated with 2 × 10⁴ conidia mL⁻¹ of Fg-IFA65_GFP; the relative amount of fungal DNA in distal tissue, as measured by qPCR at 6 dpi, was reduced in <i>CYP3</i>-dsRNA-treated leaves. Bars represent mean values ± SDs of three independent experiments. The reduction of fungal growth on <i>CYP3</i>-dsRNA-sprayed leaves was statistically significant (**P < 0.01; Student´s t test). <b>(B)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts at 6 dpi in distal leaf areas. Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in leaves sprayed with <i>CYP3</i>-dsRNA compared with <i>GFP</i>-dsRNA-sprayed controls was statistically significant (**P < 0.01, ***P < 0.001; Student´s t test). <b>(C,D)</b> Microscopy of fungal growth at semi-systemic sites of drop-inoculation with Fg-IFA65_GFP. <b>(C)</b> Successful fungal colonization (green) on TE-sprayed leaves. Profuse hyphal growth is seen inside the cells (plasma membrane stained with RH414 is highlighted in red) <b>(D)</b> Hyphal formation is strongly reduced and confined to the inoculated leaf area on <i>CYP3</i>-dsRNA-sprayed leaves. Impaired spore germination was observed in the area around the inoculation site while the surrounding cells remained free of colonization. (IF, infection hyphae; GS, germinating spore). Photographs for C and D were taken at 6 dpi.</p

(A-C) Spray-induced gene silencing (SIGS) of <i>GFP</i> expression in <i>Fusarium graminearum</i> strain Fg-IFA65_GFP.

<p>Detached second leaves of three-week-old barley plants were locally sprayed with Tris-EDTA (TE, <b>A</b>, control) or <i>GFP</i>-dsRNA <b>(B)</b>. Forty-eight hours after spraying, distal, non-sprayed leaf segments were drop-inoculated with Fg-IFA65_GFP (20 μL of a solution containing 2 x 10⁴ conidia mL^-1). <i>GFP</i> silencing efficiency was visualized 6 dpi using confocal microscopy. <b>(C)</b> <i>GFP</i> transcripts were quantified by qPCR at 6 dpi. The reduction in fungal <i>GFP</i> expression on leaves sprayed with <i>GFP</i>-dsRNA and infected with Fg-IFA65_GFP compared with TE-sprayed controls was statistically significant (***P < 0.001; Student´s <i>t</i> test). Bars represent mean values ± SDs of three independent experiments. Scale bars represent 100 μm.</p

(A-J) Confocal laser scanning microscopy of ATTO 488-labeled <i>CYP3</i>-dsRNA_A488 in locally sprayed barley leaves.

<p><b>(A-C)</b> Detection of <i>CYP3</i>-dsRNA_A488 (green) in xylem vessels of vascular bundles 24 h after spraying. <b>(D-G)</b> Longitudinal sections reveal uptake of <i>CYP3</i>-dsRNA_A488 by cells of the phloem tissue at 24 h after spraying. SE, sieve element; CC, companion cell; SP, sieve plate; PPC, phloem parenchyma cell; MC, mesophyll cell. The red cells result from the autofluorescence of chloroplasts (F,G). <b>(H-J)</b> Leaf hair cells (trichome), stomata, germinating spores (GS) and fungal hyphae strongly accumulated <i>CYP3</i>-dsRNA_A488. Fungal hyphae (IF) are stained with chitin-specific dye WGA-Alexa Fluor 594 (red) 24 h after inoculation. EC, epidermal cells. RNA signals in germinated conidia are marked by arrow heads. Scale bars 100 μm (A-H), 20 μm (F), and 10 μm (J).</p