Tillers at anthesis (+/- two days) on each plant were tagged prior to inoculation. Flag leaves of each tiller were individually inoculated on the adaxial face either with Soltrol oil alone or with a suspension of P. triticina race 1 (BBB) spores in Soltrol oil (18 mg spores ml-1 oil). Inoculations employed two passes across the leaf surface with an atomizer nozzle. Spore densities applied to leaves were determined by inoculating 6 microscope slides during the course of the inoculations and then counting the number of spores in a defined area (2.67 mm2) on each slide under a microscope. Using this method, the average spore density on inoculated leaf surfaces was determined to be 592 spores cm-1. Spore germination was assessed by spraying water agar plates lightly with inoculum and then determining the percentage of spores that had germinated after 4 h. By this method, 75% of spores had germinated (germination tube evident), while a significant portion of the other spores were oblong in shape, indicating that they were in the early process of germinating. After allowing oil to dry off of leaf surfaces for 1.5 h, pots were placed in mist chambers (one rep per chamber) and subjected to 30 min initial misting. A 30 min mist/4 min no mist cycle was then employed for three h, followed by a mist cycle of 10 min on/10 min off for 5 h, and finally a mist regime of 6 min on/10 min off for 10 h 45 min. The temperature in the mist chamber during the mist period was 21ºC +/- 1ºC. Plant were then removed from the mist chambers and allowed to dry, and they were placed back into the growth chamber.
Seed of each genotype were stratified on wet filter paper for 4 days, and then planted three to a pot in 6 inch round plastic pots (Belden Plastics, Roseville, MN) filled with a potting mixture of field soil, compost, and sand. 20 grams of Nutricote 100 day slow release 13-13-13 pellets with micronutrients (SunGro, Vancouver, British Columbia, Canada) was placed into each pot. Pots were placed in a growth chamber (Conviron PGW36, Conviron, Winnipeg, Canada) initially set at 18°C day 16°C night, with 16 h day/8 h night light regime. The pots were laid out in a split plot with three reps. Genotypes were the main effect, and the subplots were treatments (mock-inoculated or pathogen-inoculated). For each rep, 6 pots (3 plants per pot) were planted per genotype. Pots were watered on an as-needed basis. After one month, the temperature was dropped to 15°C day/13°C night temperature to help ensure synchronized flowering, and subsequently the temperature was gradually stepped up to 20°C day/17°C night temperatures (16°C day/14°C night 9 days after setting to 15°C day/13°C night, 17°C/15°C two days later, 18°C/16°C two days later, and 20°C/17°C 9 days later). Pots were also watered thoroughly with a fertilizer solution (1.86 g L-1 Peters 20-20-20 soluble fertilizer) three weeks and 8 weeks after planting, and with a more dilute solution (0.62 g L-1) of the same soluble fertilizer 6 weeks after planting. Two months after sowing, plants were ready for inoculation.
Trizol extraction of total RNA was performed according to the manufacturer's instructions, treated with RNase-free DNase, and further purified using RNeasy columns (Promega, Madison, WI) according to manufacturer's instructions.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 μg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Following fragmentation, 15 μg of cRNA were hybridized for 16 hr at 45ºC 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.
Data analysis was conducted using the Expressionist software version Pro 3.1 (Genedata AG, CH-4016 Basel, Switzerland). The probe set values from all experiments were condensed together using the MAS 5.0 algorithm (Affymetrix, I. Statistical Algorithms Description Document. 2002.). The MAS 5.0 signal data were normalized to a median target intensity of 500 as follows: P. triticina- and mock-inoculated data were normalized together for each genotype at each timepoint for a total of six comparisons. The experiment was a randomized complete block design with three replicates for each genotype, and employed univariate analysis (t-tests) between mock- and P. triticina-inoculated plants within each genotype at each timepoint, for a total of six comparisons across the entire experiment.