Ryu, C-M., Murphy, J.F., Mysore, K.S. and Klopper, J.W.
Notes: Total RNA was isolated from leaf tissue from Arabidopsis plants treated with rhizobacteria Serattia marcescens strain 90-166, 300μM benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 100μM jasmonic acid (JA), or water for 2 days. Reverse transcription reactions were performed on 1–5μg of total RNA using 200 units of reverse transcriptase, 500ng oligo d(T)12–16mer primer and 500μM dNTPs in a final volume of 20μl. Semi-quantitative RT-PCR was performed in a final volume of 75μl using 1.5μl of cDNA, 1X PCR buffer (with 1.5 mM MgCl2), 200μM dNTP, 200nM of each gene specific primer (primers were designed from the Arabidopsis PR-1 gene) and 1.5 units of GoTaq® DNA Polymerase. To ensure that only host genes and not the viral RNA transcripts were amplified, the RT reactions were performed using oligo d(T) primers. As a loading control, parallel reactions using elongation factor primers were performed. PCR conditions for all genes were as follows: One cycle of 4 minutes at 94°C, 30 seconds at 55°C and 30 seconds at 72°C was followed by 34 cycles of 30 seconds at 92°C, 30 seconds at 59°C and 30 seconds at 72°C. A 5μl aliquot was removed from each reaction after 25, 30, and 35 cycles. These aliquots were analyzed on a 1% agarose gel stained with ethidium bromide. (3371)