Transcription factors involved in basal immunity in mammals and plants interact with the same cis- regulatory elements

Kürzlich entdeckte neue microbe-associated molecular pattern (MAMP)-responsive cis-Sequenzen enthalten die Kernsequenz GACTTT, welche als WT-Box bezeichnet wird. Abhängig vom Nukleotid vor der Kernsequenz wird die WT-Box in zwei unterschiedliche Typen unterteilt. Die Typ I WT-Box mit der Sequenz TGACTTTT und CGACTTTT interagiert mit WRKY Transkriptionsfaktoren, hingegen konnte bisher kein interagierendes Protein mit den Typ II WT-Boxen GGACTTTC, GGACTTTT und GGACTTTG identifiziert werden. Die Typ II WT-Boxen sind in Arabidopsis thaliana Protoplasten für eine MAMP-Responsivität notwendig. Um mögliche Typ II WT-Box interagierende Transkriptionsfaktoren zu identifizieren, wurden bioinformatische Analysen durchgeführt. Interessanterweise wird der Transkriptionsfaktor NF-kB p65 aus Maus (Mus musculus) als Typ II WT-Box-bindendes Protein vorhergesagt. Ähnlich wie WRKY Faktoren in Pflanzen spielt NF-kB p65 eine große Rolle in der basalen Immunität in Tieren. Aufgrund dessen wurde die Interaktion von NF-kB p65 mit den Typ II WT-Boxen experimentell analysiert. NF-kB p65 benötigt die Typ II WT-Boxen GGACTTTC, GGACTTTT und GGACTTTG zur Aktivierung der Reportergenexpression in pflanzlichen Zellen. Des Weiteren benötigt NF-kB p65 die Typ II WT-Box ähnliche Sequenz aus dem pathogenesis related 1 Promotor zur Aktivierung der Reportergenaktivität in Pflanzenzellen. In vitro benötigt NF-kB p65 die WT-Box für eine direkte Bindung. In Yeast One-Hybrid Screens mit einem Typ II WT-Box enthaltenden cis-regulatorischen Modul aus dem WRKY30 Promotor wurden die WRKY Faktoren WRKY26, 40 und 41 selektiert. In pflanzlichen Zellen reprimieren WRKY26, 40 und 41 die MAMP-responsive Reportergenexpression. Um den Einfluss der WT-Box auf die von WRKY26 und 41 vermittelte Genexpression zu ermitteln, wurden beide Transkriptionsfaktoren in Aktivatoren durch Hinzufügen der GAL4 Aktivierungsdomäne (AD) umgewandelt. WRKY26AD benötigt die WT-Box zur vollen Aktivierung der Reportergenaktivität in Pflanzenzellen. In vitro bindet WRKY26, wie NF-kB p65, an die Typ II WT-Box GGACTTTC. Damit konnte zum ersten Mal gezeigt werden, dass tierische und pflanzliche Transkriptionsfaktoren, die beide in der basalen Immunität beteiligt sind, an die gleiche cis-Sequenz binden. Zusammenfassend zeigen die Ergebnisse dieser Arbeit eine Ähnlichkeit der basalen Immunität zwischen Tieren und Pflanzen in den konservierten MAMP-responsiven cis-Sequenzen und in der Erkennung der NF-kB p65 und WRKY Bindestelle.Novel microbe-associated molecular pattern (MAMP)-responsive cis-sequences harbor the core sequence GACTTT, designated WT-box. Dependent on the nucleotide in front of the core sequence, the WT-box is divided into two types. While type I WT-boxes like TGACTTTT and CGACTTTT interact with WRKY transcription factors, direct binding of transcription factors to the type II WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG has not been shown. Type II WT-boxes are required for MAMP-responsive gene expression in Arabidopsis thaliana protoplasts. To propose which kind of transcription factors may interact with these type II WT-boxes, a bioinformatic approach was employed. Surprisingly, this analysis predicts mouse (Mus musculus) NF-kB p65 as a transcription factor interacting with type II WT-boxes. Like WRKY factors in plants, NF-kB p65 plays a role in innate immunity in mammals. Therefore, the interaction of NF-kB p65 with type II WT-boxes was tested experimentally. NF-kB p65 requires the type II WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG for activating reporter gene expression in plant cells. Furthermore, the type II WT-box in the pathogenesis related 1 promoter is needed for NF-kB p65 activated reporter gene expression in plant cells. NF-kB p65 requires the WT-box for direct binding in vitro. Yeast One-Hybrid screens using a type II WT-box containing cis-sequence from the WRKY30 promoter select the transcription factors WRKY26, 40, and 41. In plant cells, WRKY26, 40, and 41 act as repressors of MAMP-responsive gene expression. To investigate whether the WT-box is required for WRKY26 and 41 mediated gene expression, both were converted into transcriptional activators by adding the GAL4 activating domain (AD). WRKY26AD requires the WT-box for activating full reporter gene expression in plant cells. In vitro WRKY26, like NF-kB p65, binds to the type II WT-box GGACTTTC. For the first time it could be shown that plant and animal transcription factors, involved in innate immunity, directly bind to the same cis-sequence. In summary, these results show similarity in innate immunity between mammals and plants in NF-kB p65 and WRKY26 binding site recognition

Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter

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.</p