Seeds of S. gesnerioides races SG3 and SG4z were surface sterilized and pre-conditioned for 9 days as previously described [13]. The germination of Striga seeds was triggered using root exudates from cowpea cultivar B301. The roots of each cowpea seedling were inoculated with 15 mg of pre-germinated S. gesnerioides using a paintbrush.
Growth protocol
Cowpea cultivar B301 seeds were surface-sterilized with 1% hypochlorite for 5 min. They were then placed between two sheets of moist glass fiber filter paper (GF/A Whatman, Piscataway, NJ), held between two blocks of moistened rockwool (Grodan Inc., Milton, ON) for 5 days and then transferred to a growth chamber, which consists of a 24cm x 24cm x 3cm Petri dish containing rockwool with a 100 μm mesh separating the cowpea roots from the rockwool [14]. The chamber was covered with aluminum foil to keep light away from the roots. Cowpea seedlings were grown for another 7 days in a controlled environment growth room under a 12 H light-dark photoperiod at 30o C.
Extracted molecule
total RNA
Extraction protocol
Root material was collected 6 and 13 days post-inoculation (dpi). The infected areas on the roots were cut (±0.5 around the infection site) and rinsed in distilled water to remove the attached Striga seeds and Striga seedlings. Control roots were cut into 1cm pieces and collected in the same manner. Root material was collected from three cowpea plants per replicate and each treatment was done in triplicate. Samples were frozen using liquid nitrogen and stored in a -80o C freezer. Total RNA was isolated from frozen, finely ground root tissues. RNA samples were treated with DNase (Roche Applied Science, Indianapolis, IN) and repurified using Qiagen RNeasy kit (Qiagen, Valencia, CA) according to the manufacturers’ instructions. RNA concentration and quality was determined using an Experion (Bio-Rad, Hercules, CA).
Label
Cy3
Label protocol
A 385,000 feature microarray was fabricated (Nimblegen Inc., Madison, WI) using 60-nucleotide long oligonucleotide synthesized based upon the nucleotide sequence of 43,253 cowpea unigenes of known and unknown function previously identified in a gene-space sequencing study. On the fabricated microarray, each predicted gene coding region is represented by six to eight 60-mer oligonucleotide probes.
Hybridization protocol
For hybridization, cDNA was prepared according to the protocol provided by Roche (Roche Applied Science, Indianapolis, IN). cDNA quality control and hybridization to the 385K arrays were performed at Nimblegen.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Data processing
Statistical analysis of Nimblegen microarray data was performed using software from the Bioconductor project [39] and implemented in the R environment for statistical computing and graphics (R Development Core Team, 2009), version 2.10.1. RMA background subtraction and normalization were applied to the microarray set. The experiment was performed using three biological replicates.
Global changes in gene expression during compatible and incompatible interactions of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides
Data table header descriptions
ID_REF
VALUE
RMA background-subtracted, normalized average contig intensity
