ABSTRACT: Transcript profiling of Bln1 silenced plants (BSMV-VIGS) relative to empty vector and buffer treated controls in barley-powdery mildew interactions
Project description:Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) was used to identify significant new genes in the regulation of host innate immunity. This experiment was designed to uncover significant changes in Bln1 (Contig12219_at)-silenced plants relative to empty vector and buffer treated controls. Five independent biological replications of a split-plot experimental design were conducted with replications as blocks, treatment with Blumeria graminis f. sp. hordei (Bgh) as the whole-plot factor, and all combinations of genotype (Mla13 and Mla9) and VIGS treatment [Buffer control (mock), BSMV:00 (empty vector), and BSMV:Bln1248] as the split-plot factor for a total of 60 GeneChip hybridizations. Ten seedlings were used as a split-plot experimental unit for each combination of replication, Bgh treatment, genotype, and VIGS treatment. Plants were grown in a controlled 20°C glasshouse prior to VIGS treatment. Twelve days after VIGS treatment, half of the plants in each replication were challenged with the compatible Bgh isolate 5874. Top halves of 5 of the 10 seedling third leaves (about 10 cm) from each split-plot experimental unit were harvested into liquid N2 at 32 hours after inoculation (HAI) - the timepoint with the highest differential Bln1 transcript accumulation (Meng et al. 2009), and after initial establishment of the perihaustorial interface (Caldo et al. 2004). The remaining 5 leaves were used to record infection phenotype 7 days later. RNA was isolated for GeneChip hybridization from the 32-HAI samples. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Yan Meng. The equivalent experiment is BB101 at PLEXdb.]
Project description:Blumeria graminis f.sp. hordei is an obligate biotrohic fungal pathogen causing powdery mildew in barley. As for other biotrophic fungi, haustorial structures are at the centre of the biotrophic interaction and molecular exchanges, delivering fungal effectors or virulence factors, and taking nutrient from the host. Haustoria are originiated by the fungus, following successful penetration of the initial penetration peg through the plant cell call. Haustorial structures mainly of fungal origin, but they are surrounding by a plant component, the extrauhaustorial membrane and matrix (EHM and EHMx) forming the extrahuastorial complex (EHMc). The plant protein make-up of the plant extrahaustorial components remained unexplored, and this is a first study trying to describe plant proteome associated with haustoria using samples enriched for these structures. Therefore, proteomes of haustoria enriched samples from the epidermis of barley leaves infected with Blumeria graminins f.sp. hordei, the causing agent of barley powdery mildew, were compared to infected epidermis and un-infected epidermis to identify haustoria associated plant proteins. Haustoria were enriched from infected epidermis by digesting epidermal cell walls with cell wall degrading enzymes prior to enrichment for haustorial structures. Proteins identified in these samples were compared to infected and uninfected epidermis samples using a non-targeted label free semi-quantitation method.
Project description:Purpose: The powdery mildew fungus, Blumeria graminis, is an obligate biotrophic pathogen of cereals and has significant impact on food security (Dean et al., 2012). B. graminis f. sp. hordei (Bgh) is the causal agent of powdery mildew on barley (Hordeum vulgare L.). We sought to address the temporal regulation of membrane trafficking associated gene expression in barley-powdery mildew interactions. We created an isogenic panel of immune signaling mutants to address three main questions: (i) which Blumeria secreted proteins are differentially regulated in response to different compromised genotypes, (ii) which barley membrane trafficking genes are altered in response to pathogen attack, and (iii) how are these genes interacting across genotypes and infection stages.
Project description:Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non-host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non-host and host powdery mildews relative to non-inoculated control plants accounted for two-thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh, suggesting a shift in resource allocation from growth to defence. A number of defence-associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti-microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non-host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway.
Project description:Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non-host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non-host and host powdery mildews relative to non-inoculated control plants accounted for two-thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh, suggesting a shift in resource allocation from growth to defence. A number of defence-associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti-microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non-host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs
Project description:Purpose: The powdery mildew fungus, Blumeria graminis, is an obligate biotrophic pathogen of cereals and has significant impact on food security (Dean et al., 2012. Molecular Plant Pathology 13 (4): 414-430. DOI: 10.1111/j.1364-3703.2011.00783.x). Blumeria graminis f. sp. hordei (Bgh) is the causal agent of powdery mildew on barley (Hordeum vulgare L.). We sought to discover novel transcripts expressed following barley infection with blumeria.
Project description:VIGS (virus-induced gene silencing) was used to silence a putative LRR-RLK, Affy ID Rbaal9i05_at, in barley. Expression patterns in leaves were compared between untreated control leaves, virus treated leaves, VIGS-Rbaal knockdown leaves and VIGS-Rbaal/PDS (phytoene desaturase) co-silenced leaves. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, David L Parrott. The equivalent experiment is BB108 at PLEXdb.] treatment: Untreated Control(3-replications); treatment: Empty Virus (no inserted plant sequences)(3-replications); treatment: Rbaal VIGS(3-replications); treatment: Co-silencing (Rbaal + PDS)(3-replications)
Project description:Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non-host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non-host and host powdery mildews relative to non-inoculated control plants accounted for two-thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh, suggesting a shift in resource allocation from growth to defence. A number of defence-associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti-microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non-host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Computed
Project description:We performed RNA-sequencing of Bgh-infected barley leaves at two different time-points after infection to examine gene expression in the barley powdery mildew isolate DH14 during plant pathogenesis.
Project description:Purpose: The powdery mildew fungus, Blumeria graminis, is an obligate biotrophic pathogen of cereals and has significant impact on food security (Dean et al., 2012. Molecular Plant Pathology 13 (4): 414-430. DOI: 10.1111/j.1364-3703.2011.00783.x). Blumeria graminis f. sp. hordei (Bgh) is the causal agent of powdery mildew on barley (Hordeum vulgare L.). We sought to identify small RNAs (sRNAs) from both barley and Bgh that regulate gene expression both within species and cross-kingdom.