Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25 000 fragments estimated to represent about 17 000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissu

Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites

Blumeria graminis f.sp. tritici, the causal agent of powdery mildew in wheat, is an obligate biotrophic fungus that exclusively invades epidermal cells. As previously shown, spraying of a solution of syringolin A, a circular peptide derivative secreted by the phytopathogenic bacterium Pseudomonas syringae pv. syringae, triggers hypersensitive cell death at infection sites in powdery mildew infected wheat. Thus, the fungus is essentially eradicated. Here we show that syringolin A also triggers hypersensitive cell death in Arabidopsis infected with the powdery mildew fungus Erysiphe cichoracearum. To monitor transcriptional changes associated with this effect, we cloned 307 cDNA clones representing 158 unigenes from powdery mildew infected, syringolin A sprayed wheat leaves by a suppression subtractive hybridization cloning procedure. These cDNAs were microarrayed onto glass slides together with 1088 cDNA-AFLP clones from powdery mildew-infected wheat. Microarray hybridization experiments were performed with probes derived from leaves, epidermal tissue, and mesophyll preparations of mildewed or uninfected wheat plants after syringolin A or control treatment. Similar experiments were performed in Arabidopsis using the Affymetrix ATH1 whole genome GeneChip. The results indicate a conserved mode of action of syringolin A as similar gene groups are induced in both species. Prominent groups include genes associated with the proteasomal degradation pathway, mitochondrial and other heat shock genes, genes involved in mitochondrial alternative electron pathways, and genes encoding glycolytic and fermentative enzymes. Surprisingly, in both species the observed transcriptional response to syringolin A was considerably weaker in infected plants as compared to uninfected plants. The results lead to the working hypothesis that cell death observed at infection sites may result from a parasite-induced suppression of the transcriptional response and thus to insufficient production of protective proteins necessary for the recovery of these cells from whatever insult is imposed by syringolin

Analysis of epidermis and mesophyll-specific transcript accumulation after syringolin A application in powdery mildew-inoculated wheat leaves

Im Gegensatz zu anfälligen Pflanzen reagieren viele resistente Pflanzen auf Pathogenbefall mit der Induktion von hypersensitiver Reaktion (HR), einer zentralen Komponente des pflanzlichen Abwehrmechanismus. Kürzlich wurde gezeigt, dass die HR in mehltaubefallenen Epidermiszellen von Weizen durch die Einwirkung von Syringolin, einem zyklischen Tetrapeptid aus Pseudomonas syringae pv. syringae, reaktiviert werden kann. In der vorliegenden Arbeit wurden mittels cDNA-Subtraktion differenziell regulierte pflanzliche Gene identifiziert und anschliessend mit Hilfe von cDNA-Mikroarray untersucht. Dabei wurde die transkriptionelle Regulation durch Einwirkung von Syringolin von insgesamt 124 Genen bestätigt. Deren mutmassliche kodierte Proteine zeigten Ähnlichkeit zu Proteinen unterschiedlichster biologischer Aktivität. Interessanterweise waren alle differenziell regulierten Gene auch durch die alleinige Einwirkung von Syringolin reguliert. Keines davon zeigte epidermisspezifisch regulierte Transkription, obschon die syringolininduzierte HR ausschliesslich in der Epidermis stattfindet. Verglichen zur pflanzlichen Antwort auf das Kontaktfungizid Cyprodinil wurde lediglich geringe Überlappung in der Gentranskription festgestellt. Silencing hervorgerufen durch transient induzierte RNA-Interferenz zeigte bei einer Auswahl von 30 Genen keine Auswirkungen auf die Pm3b-resistenzgenassoziierte HR. Anhand weiterführender Mikroarrayuntersuchungen mit unterschiedlichen Mehltauisolaten wurden jedoch 2 mutmassliche HR-Markergene unbekannter molekularer Funktion identifiziert. Eine umfassende Analyse führte zu einer neuen Hypothese, wonach Syringolin als Virulenzfaktor wirkt, welcher die Wirtsabwehr sowie HR zu unterdrücken vermag. Upon infection with pathogens, resistant plants unlike susceptible ones react with the induction of a hypersensitive response (HR) corresponding to a central component of the plant defense mechanism. It was recently discovered, that HR can be re-induced in powdery mildew-infected wheat epidermal cells by the action of syringolin, a cyclic tetrapeptide secreted by Pseudomonas syringae pv. syringae. In the present work, differentially regulated plant genes were identified by cDNA subtraction and further characterized by cDNA microarray technology. By this means, syringolin-mediated transcriptional regulation of 124 genes was confirmed. Putative encoded proteins showed similarities to proteins of a broad range of biological activity. Interestingly, all differentially regulated genes also were regulated by the action of syringolin alone. Despite the fact that the HR induced by syringolin exclusively occurs in the epidermis, none of the genes exhibited epidermis-specific transcriptional regulation. Furthermore, only marginal overlaps of gene transcription were found upon comparison between the plant response to syringolin and to the contact fungicide cyprodinil. In a selection of 30 genes, silencing provoked by transiently induced RNA interference did not have any impact on the Pm3b resistance gene associated HR. Continuative investigations by cDNA microarray using different powdery mildew isolates resulted in the identification of 2 putative HR marker genes of unknown molecular function. Comprehensive analyses lead to a novel hypothesis according to which syringolin acts as a virulence factor, capable of suppressing host defense and induction of HR in wheat

Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites.

Blumeria graminis f.sp. tritici, the causal agent of powdery mildew in wheat, is an obligate biotrophic fungus that exclusively invades epidermal cells. As previously shown, spraying of a solution of syringolin A, a circular peptide derivative secreted by the phytopathogenic bacterium Pseudomonas syringae pv. syringae, triggers hypersensitive cell death at infection sites in powdery mildew infected wheat. Thus, the fungus is essentially eradicated. Here we show that syringolin A also triggers hypersensitive cell death in Arabidopsis infected with the powdery mildew fungus Erysiphe cichoracearum. To monitor transcriptional changes associated with this effect, we cloned 307 cDNA clones representing 158 unigenes from powdery mildew infected, syringolin A sprayed wheat leaves by a suppression subtractive hybridization cloning procedure. These cDNAs were microarrayed onto glass slides together with 1088 cDNA-AFLP clones from powdery mildew-infected wheat. Microarray hybridization experiments were performed with probes derived from leaves, epidermal tissue, and mesophyll preparations of mildewed or uninfected wheat plants after syringolin A or control treatment. Similar experiments were performed in Arabidopsis using the Affymetrix ATH1 whole genome GeneChip. The results indicate a conserved mode of action of syringolin A as similar gene groups are induced in both species. Prominent groups include genes associated with the proteasomal degradation pathway, mitochondrial and other heat shock genes, genes involved in mitochondrial alternative electron pathways, and genes encoding glycolytic and fermentative enzymes. Surprisingly, in both species the observed transcriptional response to syringolin A was considerably weaker in infected plants as compared to uninfected plants. The results lead to the working hypothesis that cell death observed at infection sites may result from a parasite-induced suppression of the transcriptional response and thus to insufficient production of protective proteins necessary for the recovery of these cells from whatever insult is imposed by syringolin A

Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves.

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25,000 fragments estimated to represent about 17,000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue