title: Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.
abstract: Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene.
authors.affiliation: Institut für Genetik, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany. k.kanofsky@tu-braunschweig.de.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kanofsky
authors.affiliation: Institut für Genetik, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany.
authors.forename: Mona
authors.initials: M
authors.lastname: Lehmeyer
authors.affiliation: Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstr. 1, 38106, Braunschweig, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Schulze
authors.affiliation: Institut für Genetik, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Hehl
mesh_terms: D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D017930:Genes, Reporter; D008967:Molecular Biology / Q000379:methods*; D028528:Petroselinum / Q000235:genetics* / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000382:microbiology
keywords: MAMP; Parsley protoplasts; Plant–pathogen interaction; Synthetic promoter; Transient reporter gene assays