Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Light-dependent expression of flg22-induced defense genes in Arabidopsis.

Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes.

Chloroplast-mediated activation of plant immune signalling in Arabidopsis.

Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specific Ca(2+) signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca(2+) transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through (1)O(2)-mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.