Summary Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe‐associated molecular pattern (MAMP)‐responsive cis‐regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis‐sequence was identified which confers strong MAMP‐responsive reporter gene activity with low background activity. The 35‐bp‐long cis‐sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis‐sequence is shown to be a tripartite cis‐regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP‐responsive reporter gene expression compared to the wild‐type DJ1E promoter. The CRM consists of two WT‐boxes (GGACTTTT and GGACTTTG) and a variant of the GCC‐box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one‐hybrid screenings using a transcription factor (TF)‐only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down‐regulates MAMP‐responsive gene expression. No TFs interacting with the WT‐boxes GGACTTTT and GGACTTTG were selected in yeast one‐hybrid screenings with the TF‐only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.

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.

The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design.

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