Kazuyuki Hiratsuka

Solution structures of the trihelix DNA-binding domains of the wild-type and a phosphomimetic mutant of Arabidopsis GT-1: mechanism for an increase in DNA-binding affinity through phosphorylation.

GT‐1 is a plant transcription factor that binds to one of the cis‐acting elements, BoxII, which resides within the upstream promoter region of light‐responsive genes. GT‐1 was assumed to act as a molecular switch modulated through Ca2+‐dependent phosphorylation/dephosphorylation in response to light signals. It was shown previously that the phosphorylation of threonine 133 in the DNA‐binding domain (DBD) of GT‐1 results in enhancement of the BoxII‐binding activity. Interestingly, point mutation of Thr133 to Asp also enhances the BoxII‐binding activity. Here, we report the solution structures of hypothetical trihelix DBDs of the wild‐type (WT) and a phosphomimetic mutant (T133D) of GT‐1. First, we demonstrated that the isolated DBD of GT‐1 alone has the ability to bind to DNA, and that the T133D mutation of the isolated DBD can enhance the DNA‐binding affinity. The structures of these DBDs turned out to be almost identical. The structural topology resembles that of Myb DBDs, but all α‐helices are longer in GT‐1. Our NMR titration experiments suggested that these longer α‐helices yield an enlarged DNA‐binding surface. The phosphorylation site is located at the N‐terminus of the third α‐helix. We built a structural model of the T133D DBD:BoxII complex with the program HADDOCK. The model resembles the structure of the TRF1 DBD:telomeric DNA complex. Interestingly, the model implies that the phosphorylated side chain may directly interact with the bases of DNA. On the basis of our findings, we propose a mechanism by which the DNA‐binding activity toward BoxII of the phosphorylated GT‐1 could be enhanced. Proteins 2010.

Transient Assay System for the Analysis of PR-1a Gene Promoter in Tobacco BY-2 Cells

In order to develop a rapid and versatile assay system suitable for the analysis of regulated expression of tobacco pathogenesis-related protein 1a (PR-1a) gene, we investigated the use of the transient gene expression system in tobacco BY-2 cells by microprojectile bombardment. Using dual luciferase assay as a reporter gene expression detection system, we observed significant induction of PR-1a promoter activity by salicylic acid (SA) treatment. On the other hand, treatment with 4-hydroxybenzoic acid (4-HBA) resulted in no detectable increase in luciferase activity. Co-expression of a trans-acting factor, the NPR1/NIM1 protein of Arabidopsis, resulted in the induction of higher expression levels of the PR-1a promoter. These results suggest that the assay system is applicable for the analysis of factors involved in the regulated expression of SA-inducible defense-related genes.

Peanut stunt virus 2b cistron plays a role in viral local and systemic accumulation and virulence in Nicotiana benthamiana

To analyze the role of the 2b protein (2bP) of Peanut stunt virus (PSV) in the viral infection cycle, we constructed PSV mutants that express either no 2bP or N-terminal-truncated 2bP. The accumulation of wild-type and mutant viruses in tobacco protoplasts indicated that the 2b cistron is not essential for viral replication. Viral accumulation in Nicotiana benthamiana plants suggested that the 2b cistron is responsible for viral accumulation in inoculated and upper leaves and has a role in virulence. The involvement of eight N-terminal amino acids of 2bP in these functions is discussed.

Light regulated transcription in higher plants

Studies on the function of plant promoters have demonstrated the presence of regulatorycis-acting elements that mediate developmental or environmental signals. Analysis of many light-responsive genes showed thatcis-acting elements responsible for light regulated transcription are located within the 5′ upstream region. Numerous light responsivecis-acting elements andtrans-acting factors have been identified and characterized. The present article reviews the recent advances in studies of light regulated transcriptional regulation and signal transduction.

Evaluation of the Use of the Tobacco PR-1a Promoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.

Dmc1 fluorescent foci in prophase I nuclei of diploid, triploid and hybrid lilies

Abstract. We examined the distribution of meiotic epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that retention of the foci until the pachytene stage requires a successful homology check and synaptonemal complex (SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to pachytene, and the triploid pachytene values are similar to the pachytene values of the diploid. The species-hybrid lacks chromosome homology, has reduced SC formation and few reciprocal genetic exchanges. In this species-hybrid the number of foci at early zygotene is similar to that in the normal diploid but is dramatically reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that SC formation is reduced. In contrast the extent of the reduction in reciprocal genetic exchange in the species-hybrid is much greater than the reduction in the number of foci. We conclude that Dmc1 protein is involved in homology checking, but the impact of failure to find homology affects SC formation and reciprocal genetic exchange differentially.

Mutational analysis of the signal for a nuclear localization of proteins which accumulate specifically during meiosis in lily microsporocytes

Abstract LIM5 and LIM13 are novel meiosis-associated genes isolated from Lilium longiflorum. The presence of a hydrophobic N-terminal region predicted from the amino acid sequence has suggested that they function as extracellular structural components. However, both proteins also contain clusters of basic amino acids which may function as nuclear localization signals. To investigate the cellular localization of the protein, we tagged the C-termini of LIM5 and LIM13 with a green fluorescent protein. Transient expression of fusion proteins in onion epidermal cells revealed nuclear localization activity of both proteins. Mutational analysis indicated that amino acid sequences that constitute bipartite-type nuclear localization signals are necessary and sufficient for the intracellular localization of both proteins.