The signaling pathway mediated by salicylic acid (SA) plays a crucial role in the defense responses of plants. However, little is known about the SA-signaling pathway in monocots. To identify new components of the pathway or regulatory components that indirectly modulate the pathway in rice, we screened for SA hypersensitivity in rice FOX Arabidopsis lines. Some hypersensitive lines may express components that regulate the SA-signaling pathway either upstream or downstream of SA. Others may express SA-metabolizing enzymes or regulatory proteins involved in other signaling pathways that interact with the SA pathway.
To select SA-sensitive FOX lines, we sowed 10 sterilized T2 seeds of each line on MS medium containing 50 µM SA. Wild-type (Col-0) and SA-hypersensitive npr1 seeds were sown as controls. The seeds were cold-treated at 4 °C for 2 days, germinated on the SA-containing medium, and grown at 22 °C for 12-14 days under a 16-h light - 8-h dark cycle. npr1 plants were arrested at the cotyledon stage with bleached tissues, whereas wild-type plants grew normally. FOX lines showing similar phenotypes to that of npr1 were selected as SA-hypersensitive lines. In a secondary screening, we verified the SA dependence of the phenotypes by examining the growth of T2 plants on MS medium with or without 50 µM SA.
To gain insight into molecular basis of the SA hypersensitivity in each line, we characterized the expression of PR-1, a marker gene of SA pathway, in the SA-hypersensitive FOX lines with or without INA (2,6-dichloroisonicotinic acid, SA analog) treatment. We sowed T3ĦĦseeds on 1/2 MS medium containing hygromycin, transferred the hygromycin-resistant plants to MS medium with or without 100 µM INA, and collected them at 24 and 48 h. Wild-type and npr1 seeds were grown for 12 days on MS medium without hygromycin. Total RNA was isolated and analyzed for PR-1 expression by Northern analysis.