Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression.
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations at different stages of infection with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis : initiation (< 1 dpi) and maintenance of infection (> 4 dpi).
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations in map65-3 and ugt76b1 mutants, 3 days after water treatment and inoculation with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa)
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations in wild-type plants and pskr1-5 mutants, 3 days after water treatment and inoculation with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis.
Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression. The whole experiment was carried out in triplicate, with two time points (5 and 7 days post-inoculation) and three cell types (pathogen-proximal, pathogen-distal and uninfected control), totalling 18 microarrays. Transgenic Arabidopsis thaliana Col-0 with the transgene pDMR6:GFP was used for all experiments. Seedling populations were inoculated with 30,000-60,000 spores/ml of Hyaloperonospora arabidopsis strain Noks1 at 7 days old, and overground tissue sampled at 5 and 7 days post-inoculation. Protoplasts were generated from these samples according to Grønlund et al. 2012 JOVE, and sorted by fluorescence activated cell sorting into a GFP-positive (pathogen-proximal) and GFP-negative (pathogen-distal) population. As a control, uninfected seedlings were also sampled at 12 and 14 days old (equivalent age of 5 and 7 days post-inoculation), protoplasts generated and sorted to yield a GFP-negative population of uninfected control cells. Gene expression was analysed using NimbleGen 12 x 135K microarrays, designed for the TAIR10 genome. Data was normalised using the Robust Multichip Averaging algorithm.
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations in wild-type plants and clv mutants, 3 days after water treatment and inoculation with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis.
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations at different stages of infection with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis : initiation (< 1 dpi) and maintenance of infection (> 4 dpi). In two independent experiments, cotyledons from the ecotype Wassilewskija (WS) were inoculated with water, or with Hyaloperonospora arabidopsidis to establish a compatible interaction. Affymetrix ATH1 microarrays were used to profile Arabidopsis transcript accumulations at the initiation (mixed samples at 8 and 24 hours post inoculation, hpi; early stage) and maintenance (mixed samples at 4 and 6 days post inoculation; late stage) of the compatible interaction.
Project description:We used Arabidopsis full-genome microarrays to characterize plant transcript accumulations in wild-type plants and pskr1-5 mutants, 3 days after water treatment and inoculation with the biotrophic oomycete downy mildew pathogen, Hyaloperonospora arabidopsidis. In two independent experiments, cotyledons from the wild-type Wassilewskija (WS) ecotype and from the pskr1-5 mutant were treated with water, or inoculated with the H. arabidopsidis isolate Emwa1 to establish a compatible interaction. Affymetrix ATH1 microarrays were used to profile Arabidopsis transcript accumulations at 3 days after onset of treatment.
Project description:The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infect Arabidopsis by forming feeding structures called haustoria inside the host cell. Previous transcriptome analysis revealed host genes specifically induced during infection. However, whole infected tissue-derived RNA profiling may fail to capture the key transcriptional events that may occur exclusively in haustoriated host cells where the pathogen injects virulence effectors to modulate host immunity for successful accommodation. To understand the interaction between Arabidopsis and H. arabidopsidis at the cellular level, we established a new translating ribosome affinity purification (TRAP) system applicable to pathogen-responsive promoters, enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing new insights into the Arabidopsis/downy mildew interaction. We also expect that our new TRAP system could be applicable to several stimulus-specific contexts as well as other plant–pathogen interactions.