Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes.

Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.

The regulation of ZCT1, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in Catharanthus roseus.

Cys2/His2-type (C2H2) zinc finger proteins, such as ZCT1, are an important class of transcription factors involved in growth, development, and stress responses in plants. In the medicinal plant Catharanthus roseus, the zinc finger Catharanthus transcription factor (ZCT) family represses monoterpenoid indole alkaloid (MIA) biosynthetic gene expression. Here, we report the analysis of the ZCT1 promoter, which contains several hormone-responsive elements. ZCT1 is responsive to not only jasmonate, as was previously known, but is also induced by the synthetic auxin, 1-naphthalene acetic acid (1-NAA). Through promoter deletion analysis, we show that an activation sequence-1-like (as-1-like)-motif and other motifs contribute significantly to ZCT1 expression in seedlings. We also show that the activator ORCA3 does not transactivate the expression of ZCT1 in seedlings, but ZCT1 represses its own promoter, suggesting a feedback mechanism by which the expression of ZCT1 can be limited.

Identification of G-Quadruplex-Binding Inhibitors of Myc Expression through Affinity Selection-Mass Spectrometry.

The Myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive. One strategy for inhibition of Myc expression is through stabilization of the G-quadruplex (G4), a G-rich DNA secondary structure found within the Myc promoter; stabilization of G4s has been shown to halt transcription of downstream gene products. Here we used the Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry method, to identify compounds that bind to the Myc G4 out of a pool of compounds that had previously been shown to inhibit Myc expression in a reporter screen. Using an ALIS-based screen, we identified hits that bound to the Myc G4, a small subset of which bound preferentially relative to G4s from the promoters of five other genes. To determine functionality and specificity of the Myc G4-binding compounds in cell-based assays, we compared inhibition of Myc expression in cells with and without Myc G4 regulation. Several compounds inhibited Myc expression only in the Myc G4-containing line, and one compound was verified to function through Myc G4 binding. Our study demonstrates that ALIS can be used to identify selective nucleic acid-binding compounds from phenotypic screen hits, increasing the pool of drug targets beyond proteins.