Glycine max PI200492 21 days old (4-5 trifolates) grown in greenhouse in sterile soil with supplemental light 16 h days
Treatment protocol
Urediniospores were harvested from infected soybeans and kept under liquid nitrogen. Prior to inoculation, urediniospores were heat shocked at 40C fro 5 min and kept in a humid environment overnight. Spores were suspended in a 0.01% (v/v) Tween20/water solution and adjusted to a concentration of 250,000 spores ml-1 with a hemacytometer. Twenty-two day old soybeans (second trifoliate leaf stage) were treated with an atomizer with either of two isolates of P. pachyrhizi, HW94-1 or TW72-1, or water/tween solution at 2 ml per plant. Plants were placed in a dew chamber at 20ºC for up to 24 h, and then placed in the greenhouse set at 25ºC. Leaves were harvested from inoculated plants at 6, 12, 24, and 48 hours post inoculation and snap frozen in liquid nitrogen. 4-6 leaves were pooled for each sample
Growth protocol
Glycine max (Merr.) cultivar Komata (USDA germplasm accession PI200492) was seeded, grown, and maintained in a greenhouse with supplemental lighting to achieve 16 hour days
Extracted molecule
total RNA
Extraction protocol
trizol/guanidium isothiocyanate extraction (Chomczynski and Sacchi, 1987), followed by lithium chloride precipitation, DNAse treatment
Label
biotin
Label protocol
Affymetrix one cycle labeling kit following manufacturer's instructions
Hybridization protocol
45C, 60 rpms, 16 hours
Scan protocol
Affymetrix Gene Scanner 3000
Description
C2
Data processing
Only the Gma probes were used for the analysis. After removal of the non-Gma proces in the R programming environment, the resulting data set was normalized, scaled and background subtracted using RMA. We used the default options in the fitPLM fucntion in affyPLM: RMA convolution for background subtraction and quantile normalization. Data for multiple spots referring to one transcript were summarized using RMA tranformation of probe level data to gene expression level data. log2 expression values were tested for differences between Resistant and Susceptible using the moderated t statistic, and the results were filtered such that genes with a p,0.05 and and absolute fold change > 1.5 were considered significantly different.
