Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis laser microdissected cells at the powdery mildew infection site

ABSTRACT: Obligate biotrophs such as the virulent powdery mildew Golovinomyces orontii alter plant host cellular architecture, metabolism, and defense in order to acquire nutrients while suppressing cell death and senescence. G. orontii exclusively infects epidermal cells of Arabidopsis with clearly defined stages of infection. Host factors mediating the powdery mildew (PM) interaction are often expressed in the mesophyll cells underlying the infected epidermal cells. Therefore, in order to identify Arabidopsis processes and regulators mediating this PM interaction, we used UV laser microdissection to isolate cells at the PM infection site for global expression profiling. As part of this process, we optimized and/or developed novel tissue preparation, RNA extraction and amplification, and quality control protocols resulting in highly correlated biological replicate data. We focused on the growth and reproduction stage of the PM infection (5 days post infection) when the number of reproductive structures, conidiophores, can be quantified. Site-specific profiling increased our sensitivity dramatically, allowing us to identify specific processes, process components, and their putative regulators hidden in previous whole leaf global expression analyses. For example, the known cell cycle regulator MYB3R4 exhibits altered expression at the site of infection, as do a subset of cell-cycle-associated genes. Furthermore, null myb3r4 mutants exhibit enhanced resistance to PM with reduced conidiophores per colony, suggesting cell cycle control plays an important role in the PM interaction. Experiment Overall Design: Arabidopsis whole leaves from wild type Columbia-0 and enhanced disease susceptibility mutant eds16-1, a null isochorismate synthase 1 (At1g74710) mutant were harvested at 5 days after Golovinomyces orontii infection, microwave-fixed, paraffin-embedded and sectioned. Groups of epidermal and mesophyll cells (~20 cells/group) surrounding the G. orontii infected epidermal cell were cut using a Leica AS laser microdissection (LMD) system. In parallel, groups of epidermal and mesophyll cells were collected from uninfected leaves at 5 days from wild type Arabidopsis. LMD-isolated cells, whole leaf, whole leaf amplified and tissue scrape samples were used for RNA extraction and hybridization to Affymetrix Arabidopsis ATH1 microarrays. Gene expression profiles were obtained for wild type from all samples and for ics1 mutant from LMD infected samples. The experiment includes 2 biological replicates.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Mary Wildermuth

PROVIDER: E-GEOD-15680 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.

Chandran Divya D Inada Noriko N Hather Greg G Kleindt Christiane K CK Wildermuth Mary C MC

Proceedings of the National Academy of Sciences of the United States of America 20091214 1

To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana, laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in pre ...[more]

PMID: 20018666

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Project description:Salicylic acid (SA) is a critical molecule mediating plant innate immunity with an important role limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis thaliana. To investigate this later phase of the PM interaction, and the role played by SA, we performed replicated global expression profiling for wild type and SA biosynthetic mutant ics1 Arabidopsis from 0 to 7 days post infection. We found that ICS1-impacted genes comprise 3.8% of profiled genes with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T2 statistic ((Tai and Speed, 2006)). Functional analyses of T2-selected genes identified statistically significant PM-impacted processes including photosynthesis, cell wall modification, and alkaloid metabolism that are ICS1-independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also supports a role for ICS1 (SA) in iron and calcium homeostasis and identifies components of SA crosstalk with other phytohormones. Through our analysis, 39 novel PMâimpacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2, results in significantly reduced reproduction of the powdery mildew in a cell death independent manner. Though little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48 (Rancour et al., 2004; Park et al., 2007), an essential AAA-ATPase chaperone that mediates diverse cellular activities including homotypic fusion of ER and Golgi membranes, ER-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance. Experiment Overall Design: Arabidopsis whole leaves were harvested at 6h, 1 day, 3 days, 5 days and 7 days after Golovinomyces orontii infection for RNA extraction and hybridization to Affymetrix Arabidopsis ATH1 microarrays. Gene expression profiles were obtained for wild type Columbia-0 and enhanced disease susceptibility mutant eds16-1, a null isochorismate synthase 1 (At1g74710) mutant. In parallel, uninfected samples were collected at 0 hr and 7days from wild type and mutant plants. The experiment includes 4 biological replicates.

Project description:Grape powdery mildew (PM), caused by the biotrophic ascomycete Erysiphe necator, is a devastating fungal disease that affects most Vitis vinifera cultivars. We have previously identified a panel of V. vinifera accessions from Central Asia with partial resistance to PM that possess a Ren1-like local haplotype. In this study we show that in addition to the typical Ren1-associated late post-penetration resistance, these accessions display a range of different levels of disease development suggesting that alternative alleles or additional genes contribute to determining the outcome of the interaction with the pathogen. To identify potential Ren1-dependent transcriptional responses and functions associated with the different levels of resistance, we sequenced and analyzed the transcriptomes of these Central Asian accessions at two time-points of PM infection. Transcriptomes were compared to identify constitutive differences and PM-inducible responses that may underlie their disease resistant phenotype. Responses to E. necator in all resistant accessions were characterized by an early up-regulation of 13 genes, most encoding putative defense functions, and a late down-regulation of 32 genes, enriched in transcriptional regulators and protein kinases. Potential Ren1-dependent responses included a hotspot of co-regulated genes on chromosome 18. We also identified 81 genes whose expression levels and dynamics correlated with the phenotypic differences between the most resistant accessions ?Karadzhandahal?, DVIT3351.27, and O34-16 and the other genotypes. This study provides a first exploration of the functions associated with varying levels of partial resistance to PM in V. vinifera accessions that can be exploited as sources of genetic resistance in grape breeding programs. Eight varieties (7 resistant to powdery mildew, 2 of which are V. vinifera spp. sylvestris, 5 of which are V. vinifera spp. sativa) are infected with powdery mildew (E. necator) at two timepoints (1 dpi and 5 dpi) with three replicates for each timepoint for each condition (uninfected vs. infected), for a total of 96 samples

Dataset Information

Transcription profiling of Arabidopsis laser microdissected cells at the powdery mildew infection site

Publications

Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.

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