Rice Genetics IV - IRRI books - International Rice Research Institute

AOsRac1IED II III IV 214aaOsRac1 KCVTVGDGA VGKT 24OsRac1-G19V KCVTVGDVA VGKT 24 Dominant-active formOsRac1-T24N KCVTVGDGA VGKN 24 Dominant-negative formHsRac1 KCVVVGDGA VGKT 17BNormal formG19 T24OsRac1Dominant-active form(GTP-bound form)Dominant-negative form(GDP-bound form)35S pro35S proVOsRac1-G19VNOsRac1-T24NNOS terNOS terFig. 2. Construction of vector plasmids carrying constitutive active anddominant-negative OsRac1. (A) Constitutive active and dominant-negativeOsRac1 can be produced by single amino acid changes in the conservedregion I required for GTPase activity. The ED (effector domain) andII-IV regions are highly conserved between rice and human Rac genes. (B)Constitutive active and dominant-negative OsRac1 used for rice transformation.OsRac1 was fused with the CaMV 35S promoter and NOS terminatorfor constitutive expression in rice plants and cells.served H 2 O 2 production was inhibited by diphenylene iodonium (DPI), an inhibitorof the neutrophil NADPH oxidase. These findings suggest that an NADPH oxidasesimilar to the neutrophil enzyme is involved in Rac-induced H 2 O 2 production. Furthermore,DAB staining of leaf sheath cells of transgenic wild-type plants showedH 2 O 2 production, which appeared to be localized at the intercellular space.Cell death induced by the constitutive active OsRac1Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) signalsindicative of nuclear DNA cleavage were observed in the transformed sl cells but notin untransformed sl cells. Electron microscopy of the transformed sl cells indicatedthat cell death occurs in the transformed cells and that the observed cell death exhibitsa set of morphological changes found in apoptosis in mammalian cells (Shimamoto etal 1999). Furthermore, necrotic lesions were found in the leaves of transgenic wildtypeplants at a young stage. During maturation, discrete lesions developed in theleaves and they were frequently observed at the junction of the blade and the sheathof the leaves. These results indicate that the constitutive active OsRac1 can inducecell death in transgenic rice plants as well as in cultured cells.Molecular signaling in disease resistance of rice 325