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Abstract: To establish a system that facilitates the coordinate expression of multiple genes in a crop biotechnology setting, we tested the internal ribosome entry site (IRES) sequence from encephalimyocarditis virus (an animal viral polycistronic sequence) in rice plants. Three reporter genes, green fluorescent protein (Gfp), β-glucuronidase (Gus) and luciferase (Luc), were linked to IRES under the control of the ubiquitin promoter, generating FIG (G f p-I RES-G us) and FIGIL (G f p-I RES-G us-I RES-L uc) constructs, respectively. We obtained a total 38 transgenic lines using these vectors (24 for FIC and 14 for FIGIL) and the transgene integration and copy numbers for each were determined by Southern blot analysis. By northern blot analysis using Gfp, Gus and Luc probes, both the FIG and FIGIL rice plants showed multiple truncated transcripts, including full-length recombinant genes. Western blot analyses using GFP and GUS antibodies revealed the accumulation of GFP but not GUS protein in both FIG and FIGIL rice leaf tissues. Subsequent GFP and GUS reporter expression patterns showed GFP fluorescence but not GUS-staining in both FIG and FIGIL transgenic rice callus tissues. These results suggest that the direct application of animal viral IRES sequences is not sufficient to induce polycistronic expression in rice plants due to the production of multiple truncated transcripts.

Abstract: Members of the NAM-ATAF-CUC (NAC) protein family are plant-specific transcription factors that contain a highly conserved N-terminal NAC-domain and diverse C-terminal regions. They have been implicated in plant development and abiotic stress responses. To identify interacters of rice NAC-domain proteins (OsNACs), we performed yeast two-hybrid screening of rice cDNA library using OsNAC5 as a bait, and the results showed that OsNAC5 interacts with other OsNACs including itself. To delineate an interacting domain, a series of deletion constructs of four OsNACs were made and transformed into yeast in various combinations. The results revealed that the conserved NAC domain of OsNACs plays a primary role in homodimer and heterodimer formation, and a part of C-terminal sequence is also necessary for the interaction. In vitro pull-down assays using recombinant OsNAC proteins verified the dimer formations, together suggesting that OsNACs may act by forming homodimers and/or heterodimers in plants.