Transcription profiling by array of Medicago truncaluta seedling roots innoculated with Macrophomina phaseolina to study M. truncaluta and M.phaseolina interaction
ABSTRACT: This experiment was designed to study the interactions between Medicago truncatula and the charcoal rot pathogen Macrophomina phaeolina. Two-week-old plants grown in Magenta boxes supplied with 1/2 MS salt and 1% sucrose were inoculated with M. phaseolina covered wheat seeds, and roots were harvested at 24, 36 and 48 hours after inoculation. Control plants were mock inoculated with a sterile wheat seed, and roots were harvest 24 hours later. Pooled RNAs were used in the array experiment using Affymetrix GeneChip(r) Medicago Genome Array.
Project description:To identify the nonhost resistance genes, a time-series based global expression profiling was performed in wheat using Agilent gene expression 44K microarray array. Seven-day-old wheat plants of cv. Renan was inoculated with spore density of 50-80 conidia mm-2 of adapted (Bgt) or non-adapted (Bgh) powdery mildew pathogen, and the abaxial epidermis of inoculated primary leaves or from non-inoculated control leaves was peeled at 6, 12, 24 and 74 h after inoculation. Total RNA was extracted by using the RNeasy plant mini kit with on-Column DNase digestion (Qiagen, Hilden, Germany), and hybridized to a 44K Agilent oligonucleotide custom array of wheat.
Project description:The supply of soluble silicon (Si) to plants has been associated with many benefits that remain poorly explained and often contested. In this work, the effect of Si was studied on wheat plants under both control and pathogen stress (Blumeria graminis f.sp. tritici (Bgt)) conditions by conducting an exhaustive transcriptomic analysis (55,000 genes) aimed at comparing the differential response of plants under four treatments. The response to the supply of Si on control (uninfected) plants was limited to 47 genes providing little evidence of regulation of a specific metabolic process. Plants reacted to inoculation with Bgt by an up-regulation of many genes linked to stress and metabolic processes and a down-regulation of genes linked to photosynthesis. Supplying Si to inoculated plants largely prevented disease development, a phenotypic response that translated into a nearly perfect reversal of genes regulated by the effect of Bgt alone. These results suggest that Si plays a limited role on a plant’s metabolism in absence of stress, even in the case of a high-Si accumulating monocot such as wheat. On the other hand, the benefits of Si, in the form of biotic stress alleviation, were remarkably aligned with a counter-response to transcriptomic changes induced by the pathogen Bgt. Experiment Overall Design: Wheat culture in hydroponic system : Experiment Overall Design: Hydroponic systems were used to precisely control Si feeding of plants and to reduce external Si contamination. Seeds of wheat cultivar AC Drummond, chosen for its known high susceptibility to Bgt, were sown in 9-cm pots in a nylon bed consisting of nylon stockings cut into small ribbons. Hydroponic systems were set up to immerse roots for 15 minutes every 30 minutes. The plants were grown in a greenhouse (16 h light at 22°C and 8h dark at 18°C, 80% humidity). Plants were immersed in distilled water only during the first week following sowing. Experiment Overall Design: Si amendment and Bgt inoculation: Experiment Overall Design: Wheat plants were grown in the presence (Si+) or absence (Si-) of Si and inoculated (B+) or not (B-) with Bgt for a total of four treatments: Si-B-, Si+B-, Si-B+, and Si+B+. These treatments were applied in the following manner. One week after sowing, distilled water was replaced by a Hoagland solution amended or not with potassium silicate (Kasil 6, PQ Corp etc) at a concentration of 1.7 mM (Si+ Hoagland). Twice a week, Si was added to the Si+ nutrient solution in order to maintain Si concentration at 1.7 mM. Every other week, both Si- and Si+ Hoagland solutions were renewed and the pH adjusted to 5.8 in all systems at each Si addition or nutrient solution renewal. Experiment Overall Design: Four weeks after sowing (i.e. three weeks after Si amendment started), half the plants were inoculated with Bgt as described previously. Briefly, one day prior to inoculation, reservoir wheat plants heavily infected with Bgt were gently shaken to remove old spores and to stimulate the production of fresh ones. The inoculation was performed by shaking these infected wheat plants over the experimental plants . Experiment Overall Design: RNA preparation: Experiment Overall Design: Total RNA was extracted from leaves of three plants per treatment (three biological replicates) with the RNeasy plant kit (Qiagen, Hilden, Germany). Concentration and quality of RNA were assessed on a Nanodrop spectrophotometer (Nanodrop, Wilmington, De, USA) and 2100 Bioanalyzer (Agilent, Palo Alto, Ca, USA). Biotin-labeled cRNAs were synthesized using MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, Tx, USA) following the manufacturer’s instructions using 1 µg total RNA. Labelled cRNA was then fragmented using 5X Array Fragmentation Buffer supplied with the MessageAmp II-Biotin Enhanced Kit. Experiment Overall Design: Microarray hybridization: Experiment Overall Design: The labeled samples were added to an Affymetrix GeneChip® Wheat Genome Array (Affymetrix, Santa Clara, CA, USA), according to the manufacturer’s instructions. The wheat chip contains probe sets representing 55,052 transcripts for all 42 chromosomes in the wheat genome. The chips were hybridized for 16 h at 45 °C in a rotisserie oven at 60 rpm. Following hybridization, the arrays were washed and stained in an Affymetrix Fluidics Station 450, according to the standard protocol from Affymetrix, and scanned using an Affymetrix Scanner 3000. Twelve chips were prepared and corresponded to three biological replications of each of the four treatments (Si-B-, Si+B-, Si-B+, and Si+B+).
Project description:Crown rot of wheat, caused by Fusarium pseudograminearum and other Fusarium species is an important disease globally. To understand the host response to challenge by Fp, we examined gene exression changes in the stem base of the wheat variety Kennedy, following inoculation with macroconidia using the Affymetrix GeneChip Wheat Genome Array. Induced genes included mainly those with defensive functions such as genes encoding anti-microbial proteins as well as oxidative stress-related proteins, signalling molecules, and proteins involved in both primary and secondary metabolism. This study is the first comprehensive analysis of the wheat transcriptome during crown rot infection and provides new insights into the host processes involved in plant defence against this pathogen. Experiment Overall Design: There are six samples, three F. pseudograminearum inoculated samples and three mock inoculated samples. Each sample consists of 2cm of stem base from approximately 20 plants.
Project description:Recent attempts to increase endogenous disease resistance of plants by overexpression of anti-fungal transgenes have shown a potential of this method. However, it has also been shown that such improvements are usually small. One of the obvious reasons for this low anti-fungal effect might be the regulation of endogenous genes in parallel. In this project, we will study the effect of anti-fungal transgenes on the endogenous gene expression. Such effects might relate to substantial equivalence which is a biosafety issue of concern to the public. The GeneChip Wheat Genome Array will be used to detect expression of defence response genes and key genes of metabolic pathways. We will use wheat plants transformed with anti-fungal gene of specific effect against a small group of seed transmitted, pathogenic fungi (KP4 against smuts and bunts). Transformed spring wheat line will be challenged by stinking smut (inhibited by KP4). The effect on the endogenous gene expression will be tested for plants grown in the field in collaboration with the USDA Department. This work will contribute to our understanding of plant defence responses in general and may allow improving strategies to strengthen these responses. Teliospores of pathogenic races T-1, T-5 and T-16 of T. caries provided by a collection in Aberdeen, ID, USA were used for the tests. Seeds of the genetically engineered Swiss spring wheat variety Greina (GrKP4) and the null-segregant control line (Gr0) were coated with spores and Individual plants were scored for bunt symptoms. For microarray analysis only samples inoculated with T1 and T16 were used.
Project description:Medicago truncatula, which has a relatively small diploid genome, has been adopted as a model species for legume genomics. To enhance its value as a model, we have generated a gene expression atlas that provides a global view of gene expression in all major organ systems of this species, with special emphasis on nodule and seed development.
Project description:Fusarium Head Blight (FHB) is a disease of wheat and other cereal crops, where, among other species, Fusarium graminearum infects the wheat inflorescence. Microarrays were used to observe differential gene expression in FHB-challenged spikes of the two European winter wheat genotypes Dream (moderately resistant) and Lynx (susceptible). Plants were either inoculated with the Fusarium graminearum strain IFA 65 (IFA Tulln) (500 macroconidia/floret) or were as control plants mock treated with desalted water. The inocula were injected into four spikelets at early anthesis and spikelets were later on collected at 32 and 72 h after inoculation. Four plants were sampled per genotype/treatment/sampling date. Total RNA was extracted from collected spikelets, and microarray analysis was performed using the Affymetrix Wheat GeneChip.
Project description:Global transcriptome patterns were performed using ORE1-IOE-2h (2h after Estradiol and Mock treatment) as well as transiently (6h) overexpressed Arabidopsis mesophyll cell protoplasts To identify genes more rapidly responding to elevated ORE1 expression we here repeated the previous experiment (Balazadeh et al., 2010), but shortened the EST induction time to 2 h (ORE1-IOE-2h dataset). Furthermore, to exclude potential misinterpretation due to EST treatment we also included an experiment where we transiently expressed a 35S::ORE1 construct in Arabidopsis mesophyll cell protoplasts and extracted RNA 6 h after the transfection (35S::ORE1-6h dataset);
Project description:After-ripening induced seed dormancy release in wheat is associated with mRNA oxidation. We used Affymetrix GeneChip Wheat Genome Array to identify mRNAs differentially oxidized by after-ripening Dormant and after-ripened seeds in dry state were used for isolating oxidized mRNAs and hybridization on Affymetrix GeneChip. After-ripened seeds were generated by storing dormant seeds at room temperature for 10 months.