Identification and characterization of novel defence and PCD signalling components in Arabidopsis: by using syp121 syp122 as a tool
Research output: Book/Report › Ph.D. thesis › Research
Plants protect themselves from pathogens by activating a defence signalling network. The Arabidopsis double mutant syp121 syp122 is dwarfed and mimics a response as if it was attacked by pathogens. Using EMS as mutagen on syp121 syp122, a suppressor screen was performed. More than 200 partially rescued syp121 syp122 ssdx (suppressor of syntaxin-related death) lines were collected. SSD genes are typically required for pathogen defence. In this PhD project, using some of these triple mutant lines, SSD6 and SSD12 were identified to be novel genes by Mutmap and complementation test. SSD6 encode a large protein with at least six domains with predicted functions, and mutations in five of these showed that they are important for the lesion mimic phenotype of syp121 syp122. Subcellular localization showed SSD6 to function on the ER. In the project, a split-GFP Gateway vector system was developed for topology studies of membrane proteins, and SSD6 was found to be an ER membrane-anchored cytosolic protein. The position of SSD6 in the defence signalling network was studied using syp121 syp122 ssd6 ssdy quadruple mutants, which suggested that SSD6 is not involved in any known signalling pathway. All plants have homologues of SSD6, but none of these have been formally reported. Besides, there are two closely related proteins in Arabidopsis, SSH1 and SSH2, which were also studied in the project. The second gene, SSD12 was confirmed to encode PRLIP2. PRLIP2 contains a signature pentapeptide motif, GXSXG, which suggested this protein to have lipase activity. By using site-directed mutagenesis to make substitutions (S162A and S162F) of the serine residue in the GXSXG motif, we confirmed it to be essential for the function of PRLIP2. The ssd6 and prlip2 mutants were inoculated with different pathogens to test the involvement in defence. The position of PRLIP2 in the defence signalling network was studied like in the case of SSD6, and the result suggested that PRLIP2 is neither involved in any known signalling pathway.
|Publisher||Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen|
|Publication status||Published - 2016|