Redox-active cysteines in TGACG-BINDING FACTOR 1 (TGA1) do not play a role in salicylic acid or pathogen-induced expression of TGA1-regulated target genes in Arabidopsis thaliana.

Salicylic acid (SA) is an important signaling molecule of the plant immune system. In Arabidopsis thaliana, SA biosynthesis is indirectly modulated by the closely related transcription factors TGACG-BINDING FACTOR 1 and 4 (TGA1 and TGA4, respectively). They activate expression of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1, the gene product of which regulates the key SA biosynthesis gene ISOCHORISMATE SYNTHASE 1. Since TGA1 interacts with the SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) in a redox-dependent manner and since the redox state of TGA1 is altered in SA-treated plants, TGA1 was assumed to play a role in the NPR1-dependent signaling cascade. Here, we identified 193 out of 2090 SA-induced genes that require TGA1/TGA4 for maximal expression after SA treatment. One robustly TGA1/TGA4-dependent gene encodes for the SA hydroxylase DOWNY MILDEW RESISTANT 6-LIKE OXYGENASE 1, suggesting an additional regulatory role of TGA1/TGA4 in SA catabolism. Expression of TGA1/TGA4-dependent genes in mock/SA-treated or Pseudomonas-infected plants was rescued in the tga1 tga4 double mutant after introduction of a mutant genomic TGA1 fragment encoding a TGA1 protein without any cysteines. Thus, the functional significance of the observed redox modification of TGA1 in SA-treated tissues remains enigmatic.

Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.

To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.

Identification of the Components Involved in Cyclic Di-AMP Signaling in Mycoplasma pneumoniae.

Bacteria often use cyclic dinucleotides as second messengers for signal transduction. While the classical molecule c-di-GMP is involved in lifestyle selection, the functions of the more recently discovered signaling nucleotide cyclic di-AMP are less defined. For many Gram-positive bacteria, c-di-AMP is essential for growth suggesting its involvement in a key cellular function. We have analyzed c-di-AMP signaling in the genome-reduced pathogenic bacterium Mycoplasma pneumoniae. Our results demonstrate that these bacteria produce c-di-AMP, and we could identify the diadenylate cyclase CdaM (MPN244). This enzyme is the founding member of a novel family of diadenylate cyclases. Of two potential c-di-AMP degrading phosphodiesterases, only PdeM (MPN549) is active in c-di-AMP degradation, whereas NrnA (MPN140) was reported to degrade short oligoribonucleotides. As observed in other bacteria, both the c-di-AMP synthesizing and the degrading enzymes are essential for M. pneumoniae suggesting control of a major homeostatic process. To obtain more insights into the nature of this process, we have identified a c-di-AMP-binding protein from M. pneumoniae, KtrC. KtrC is the cytoplasmic regulatory subunit of the low affinity potassium transporter KtrCD. It is established that binding of c-di-AMP inhibits the KtrCD activity resulting in a limitation of potassium uptake. Our results suggest that the control of potassium homeostasis is the essential function of c-di-AMP in M. pneumoniae.