Salicylic acid (SA)-dependent signaling is a major plant defense pathway that induces pathogenesis-related (PR) genes, including PR-1. Arabidopsis NPR1 (AtNPR1) is a central regulator of this pathway and acts with TGA-family bZIP transcription factors. Here, we characterized a rice TGA-family bZIP cDNA that was initially designated OsTGA1. Re-examination of the retained full-length 1,419-bp coding sequence and deduced 472-aa sequence, together with the reported gene-specific primers, assigns the clone to TGAL1, an alternative transcript of the OsbZIP08/BZIP8 locus (RAP: Os01g0808100; MSU: LOC_Os01g59350.1/.2; representative cDNA accession: AK069158; protein accession: BAG91290.1). This assignment relates the clone to the TGAL1 sequence analyzed by Chern et al. (2014) and distinguishes it from the chromosome-5 OsTGA1/OsbZIP41 locus. Southern blotting was consistent with a single predominant genomic locus under the conditions used, and RT-PCR showed tissue-dependent transcript accumulation. A TGAL1–GFP fusion was predominantly nuclear in transgenic Arabidopsis. Yeast two-hybrid assays detected TGAL1 self-association and interaction with AtNPR1. Electrophoretic mobility shift assays showed sequence-specific binding of recombinant TGAL1 to the as-1 element of the Arabidopsis PR-1 promoter; addition of AtNPR1 increased the apparent abundance or stability of the TGAL1–DNA complex under the assay conditions. These results provide complementary molecular and biochemical characterization of TGAL1/OsbZIP08 and demonstrate cross-species biochemical compatibility with AtNPR1 in the assays performed, without establishing an endogenous NH1/OsNPR1-dependent defense mechanism in rice.

Abstract
Introduction
Materials and Methods
    1. E. coli, yeast strains and plant materials
    2. Southern blot analysis
    3. RNA isolation and RT-PCR
    4. Subcellular localization
    5. Yeast two-hybrid assay
    6. Preparation of GST- and His-tagged fusion proteins
    7. Electrophoretic mobility shift assay (EMSA)
    8. DNA sequencing, sequence comparison, andclone nomenclature
Results and Discussion
    1. Clone identity and nomenclature
    2. Sequence and predicted properties of TGAL1/OsbZIP08
    3. Genomic organization of the TGAL1/OsbZIP08 locus
    4. Tissue-dependent accumulation of TGAL1 transcripts
    5. Subcellular localization of TGAL1 in a heterologousArabidopsis system
    6. Self-association of TGAL1 in yeast
    7. Interaction between TGAL1 and Arabidopsis NPR1in yeast
    8. Binding of TGAL1 to the as-1 element of theArabidopsis PR-1 promoter
    9. Effect of Arabidopsis NPR1 on the TGAL1-DNAcomplex
References

• Min Kyoung Kim(Researcher, Division of Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Dhruba Khakurel(Researcher, Division of Life Science, Gyeongsang National University, Jinju, 52828, Korea)
• Tae Won Kim(Professor, Department of Smart Farm, Chungnam state University, Cheongyang, 3303, Korea)
• Byeong Gyun Jeon(Professor, Department of Biology Education, Gyeongsang National University, Jinju, 52828, Korea)
• Sung Ho Lee(Professor, Division of Life Science & Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Korea) Corresponding author