Chapter title |
Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System
|
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Chapter number | 11 |
Book title |
Plant Synthetic Promoters
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Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-6396-6_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6394-2, 978-1-4939-6396-6
|
Authors |
Konstantin Kanofsky, Mona Lehmeyer, Jutta Schulze, Reinhard Hehl |
Editors |
Reinhard Hehl |
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. |
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