Genotype: rre1-1 (rapid response to elicitor 1-1), a T-DNA insertion mutant (SALK_066755). RRE1 (At2g35000) enocdes a protein with a RING domain found in ubiqitin E3 ligases. Treatment: incubated with hydrolyzed crab-shell chitin for 30 minutes. Growth Conditions and Treatments: All experiments were performed as previously described (Ramonell et al., 2002, Mol. Plant. Pathol. 3(5), 301-311). Arabidopsis thaliana seeds were surface sterilized. Approximately 500 seeds (10 micrograms) were placed in 250 mL Erlenmeyer flasks containing 125 mL of liquid Murashige and Skoog medium (Sigma, St Louis, MO, USA) containing 2% dextrose. These flasks were incubated with gentle shaking for 14 days under constant illumination (125 µEinsteins m-2 s-1) at 22ºC. Acid-hydrolysed, crab-shell chitin (Sigma, St Louis, MO, USA) was added to give a final concentration 100 mg/L. The control flasks received an equivalent amount of sterile, double-distilled water for each treatment. Flasks were incubated for 30 minutes with the crab-shell chitin. Whole seedlings from each treatment were collected, the medium was drained, and the seedlings were rapidly forzen in liquid nitrogen. The results of four replicates are presented. RNA and Microarray Methods: Total RNA was extracted from the plants using a modified Trizol method (Chomczynski and Sashi, 1987) (see also Protocols Manual at the AFGC web page http://www.arabidopsis.org/info/2010_projects/comp_proj/AFGC/RevisedAFGC/AFGC_Protocols_Dec_2001L.pdf) and purified with a silica membrane column (Qiagen, RNeasy). Fifteen micrograms biotinylated complementary RNA (cRNA) was prepared and used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the Affymetrix manufacturer’s protocols. The array images were analyzed with the Affymetrix GeneChip Operating Software (GCOS) 1.1 with the target intensity set to 500. These data were imported into GeneSpring 7.0 (Silicon Genetics, Redwood City, CA, USA). To remove chip-to-chip signal variation, each measurement was divided by the 50.0th percentile of all measurements in that sample. All samples were normalized to the reference data set, consisting of four replicates of Columbia-0, untreated. Each measurement for each gene was divided by the median of that gene’s intensity in the reference data set. The normalized values (Normalized Ratio) are reported along with the intensity values for this array. Keywords = chitin, defense, elicitor, mutant, PAMP, protein degradation, ring zinc finger protein, ubiquitin E3 ligase Lot batch = 510690
As defined by Affymetrix, there are the probe set identifiers, each of which is unique to a specific probe set defining a specific reagion of a singal gene or set.
This is the final calculated measurement for each probe set idendifier that has been made comparable across all samples and rows.
A qualitative measurement indicating if the probe set is detected (Present; P), not detected (Absent; A), or marginally detected (Marginal;M)
A p-value indicating the significance of the Detection call. A Detection p-value measures the probability that the discrimination scores of all probe pairs in the probe set are above a certain level, and that the target is likely to be Present.
A normalized ratio for each probe set calculated in GeneSpring 7.0 as described under Sample Description.