Plants were heavily inoculated with the Puccinia triticina race MFBL suspended in Soltrol 170 light oil (Chevron Phillips Chemical Company, The Woodlands, TX), and were then incubated in a mist chamber with 100% RH for 12 hr. The plants were returned to the growth chamber until sampling at 48 hr after removal from the mist chamber.
Plants were grown in 4-liter pots in growth chamber (Conviron PGW-36) under 15C night/20C day (+/- 1C) with a 16 hr photoperiod. Pots were fertilized at planting with 5 g/pot 14-14-14 Osmocote (The Scotts Company, Marysville, OH) and thereafter every two weeks with 0.5 g/pot 15-30-15 Miracle-Gro All Purpose Plant Food (Scott's Miracle-Gro Products, Inc., Marysville, OH) and 0.1 ml/pot Fertilome Chelated Liquid Iron and Other Micronutrients (Voluntary Purchasing Groups, Inc., Bonham, TX).
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Following fragmentation, 15 microg of cRNA were hybridized for 16 hr at 45C on GeneChip wheat Genome Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 450.
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
The data were analyzed with Affymetrix GCOS software using default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.