Growth Conditions and Treatments: Arabidopsis thaliana L. (Columbia-0) seeds were sterilized and grown hydroponically as previously described (Zhang et al., 2002, MPMI 15(9) 963-970). Briefly, Arabidopsis seeds were surface sterilized and allocated in 50 ml Falcon tubes containing 10 ml of liquid medium (1 X Murashige and Skoog Basal Salt Mixture + 2% Dextrose, pH 5.8). The tubes were incubated with gentle shaking in a growth chamber with the following settings: constant light at 125 µmol m-2 sec-1 at a temperature of 23 °C. After 14 days, the seedlings were treated with either hydrolyzed crab-shell chitin (Sigma-Aldrich, St. Louis, Missouri) at a final concentration of 100 µg/ml or the purified chito-tetramer (degree of polymerization, d.p. = 4) or octamer (d.p. =8) at a final concentration of 1 µM. The control seedlings were similarly treated with an equivalent amount of water. Whole seedlings were collected 30 minutes after treatment, immediately frozen in liquid N2, and stored at –80 °C for later use. Three independent replicates were conducted. 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, powdery mildew, Erysiphe cichoracearum
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.
VALUE
This is the final calculated measurement for each probe set idendifier that has been made comparable across all samples and rows.
ABS_CALL
A qualitative measurement indicating if the probe set is detected (Present; P), not detected (Absent; A), or marginally detected (Marginal;M)
DETECTION P-VALUE
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.
Normalized Ratio
A normalized ratio for each probe set calculated in GeneSpring 7.0 as described under Sample Description.
