Chemical-Genetic Characterization of VICTR & RDA2 Homologous Genes in the Regulation of ABA and DFPM Signal-Transduction in Arabidopsis Plants
- Author(s): Ramirez, Eduardo
- Advisor(s): Schroeder, Julian I
- Komives, Elizabeth A
- et al.
The hormone abscisic acid (ABA) triggers signal transduction that mediates drought resistance. Through a chemical genetics approach, a synthetic small molecule “DFPM” ([5-(3,4-dichlorophenyl) furan-2-yl]-piperidine-1- ylmethanethione) was shown to stimulate plant defense related genes and negatively regulates ABA signaling. DFPM independently induces root growth arrest in Arabidopsis thaliana through the VARIATION IN COMPOUND TRIGGERRED ROOT growth response (VICTR). VICTR encodes for Toll-Interleukin 1 Receptor-nucleotide binding Leucine-rich repeat (TIR-NB-NLR) protein. DFPM-screening has led to the identification of the RESISTANCE TO DFPM-INHIBITION OF ABA SIGNALING2 (RDA2) locus, which encodes for a Lectin-Receptor Kinase (LecRK). RDA2 plays a key role in DFPM-mediated inhibition of ABA signal-transduction and phosphorylating mitogen-activated protein kinases (MAPKs) 3 & 6 (MPK3MPK6). Two genes that lie upstream of RDA2 were characterized as LecRKs and found to play a role in mannitol-induced stressed, thus naming them ENHANCED SHOOT GROWTH UNDER MANNITOL STRESS (EGM) 1 & 2 (EGM1 & EGM2). I am interested in investigating the role of VICTR and its homologous genes regulating ABA signaling-transduction after exposure to DFPM. This project also aims to understand the role of the EGM homologous genes with respect to RDA2 after exposure to DFPM. In order to study the role of VICTR and its homologous tandem genes I utilized Near Isogenic Lines (NILs) NIL-Col-0 which contains functional VICTR homologs, and NIL-Bu-5 which does not contain any VICTR homologs. I also used mutant lines of RDA2 (rda2-1 and rda2-2) as well as T-DNA lines of EGM1 & 2 (egm1 & egm2) and an artificial microRNA (amiRNA) line targeting both EGM genes (egm-amiRNA) to investigate the role of these genes in DFPM-signaling. I first grew seedlings of Col-0, NIL-Col-0, and NIL-Bu-5 for two weeks and treated the plants with various chemical treatments followed by RNA extraction, cDNA synthesis, and qRT-PCR to analyze expression of ABA reporter genes. Using lines of the same genotypes transformed with pRAB18::GFP I attempted to compare relative fluorescence after exposure to ABA and DFPM chemical treatments. I also compared DFPM-mediated MAPK-activation across various time points for Col-0 and NILs as well as the rda2, egm1 & 2, and egm-amiRNA lines. I found that the genetic region comprised VICTR and its homologous genes do not play a role in the activation of MAP Kinase in response to DFPM and are not involved in the crosstalk between ABA and DFPM-signaling transduction. RDA2 is involved in activation of MAPKs and both EGM1 and EGM2 are possibly involved in co-regulating RDA2. Further analysis and higher order mutants of VICTR and RDA2 homologous genes will allow us to understand their function in ABA and DFPM signaling-transduction.