Functional Analyses of EL5, a Ubiquitin Ligase with Anti-cell Death Function in Rice

Abstract

Rice EL5 is a membrane-localized RING-type ubiquitin ligase (E3). Transgenic rice plants overexpressing mutant EL5 proteins (mEL5) that are deficient in E3 activity showed a rootless phenotype accompanied by cell death in root primordia. Thus, we concluded that EL5 functions as an anti-cell death E3 in root (Koiwai et al, 2007). We report here that treatment with higher concentration of NO₃^-, NO₂^- or Ca²⁺ specifically caused necrosis of root tip in mEL5-expressing plants. Treatment with SNP, nitric oxide (NO) donor, also enhanced cell death in mEL5-expressing callus. These results suggest that EL5 is involved in NO stress tolerance in rice. We next searched EL5-interacting proteins. Three isoforms of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC1, GapC2, and GapC3) were pulled-down with the His-tagged C-terminal region of EL5 (A195-N325) from the extract of suspension-cultured rice cells. Analysis using the bacterial two-hybrid system strongly suggested that GapC2 interacts with the P249-S276 region of EL5.