False positives were determined by executing the beta-galactosidase assay test. Only 10 colonies did not showed the characteristic blue color 4 hours after the addition of the substrate. The re-transformation process performed on the first 30 DNA interactors yielded several false positives with no growth, most coinciding with the results from beta-galactosidase assay.
All sample cDNAs obtained from the remaining unsequenced colonies amplified at the expected WRKY1 300 base pair band except three from the Moroberekan and three from the C64 library. These six were sequenced. PCR amplification was also done on the suspected false positives identified by the beta-galactosidase and retransformation processes. All colonies amplified at the expected band, indicating that there were no false positives in this screen and that all previously suspected false positive colonies contained the WRKY1 gene.
The sequencing of the initial 17 and the remaining 6 samples yielded 3 types of interactors �VWRKY1, WRKY2, and WRKY3. WRKY1 was obtained 8 times by from the samples sequenced with 3 different cDNA fragments (or 139 times including the results from the PCR amplification test), WRKY2 was obtained four times with 2 different cDNA fragments, and WRKY3 was obtained once with 1 type of cDNA fragment. From the six colonies sequenced after PCR amplification, the three from the Moroberekan library were all WRKY2��s and the three in the C64 library were two WRKY2��s and one WRKY3. The sequence of WRKY1 is 87% similar to the DNA-binding protein alpha-Amylase Binding Factor-2 (ABF-2) in wild oat and WRKY2 was determined to be the RPR1 transcription factor in rice. WRKY3 did not match with the genomic sequence of any know rice protein but does resemble WRKY1.
After sequence analysis through online protein/DNA databases and programs (NCBI BLAST, website), the sequences of WRKY1 and WRKY2 were determined to be 37% identical and 48% similar to each other; WRKY1 is 38% identical and 47% similar to Xa21, WRKY2 is 42% identical and 52% similar to Xb10, and WRKY1 and WRKY3 are 70% similar.
Plant leaves inoculated with strains J18 (virulent on Xa21) or J18 (1078) (Avirulent on Xa21) were frozen at 0, 15��, 1, 2, 4, 6, 8, 12, and 24 hours. Data indicated that WRKY1 was upregulated (activated) almost immediately after the introduction of pathogens or wounding and disappeared at 6 hours. Xb10 was also upregulated almost immediately and disappeared at 24 hours; WRKY2 was upregulated at 15min and 1 hour, disappeared at 2hrs, and reappeared at 4hrs and finally subsides; and WRKY3 was upregulated at 0, 1, and 2 hours, disappeared at 4hrs, and reappeared at later time points.