Senescence Networking: WRKY18 is an Upstream Regulator, a Downstream Target Gene, and a Protein Interaction Partner of WRKY53

Transcriptional reprogramming is a central feature of senescence regulation, implying an essential role for transcription factors. A regulatory function has already been attributed to different members of the plant-specific NAC and WRKY families in Arabidopsis but also in other plant species. WRKY53 is one important senescence regulator of the Arabidopsis WRKY family that is tightly regulated on different levels. In this study we show that WRKY18, which was formerly characterized as a downstream target of WRKY53 in the WRKY network, also regulates the expression of WRKY53. WRKY18 is able to bind directly to different W-boxes in the WRKY53 promoter region and to repress expression of a WRKY53 promoter-driven reporter gene in a transient transformation system using Arabidopsis protoplasts. Consistent with its repressing function on WRKY53 as a positive senescence regulator, WRKY18 overexpression led to delayed senescence, whereas wrky18 mutant plants exhibited a clearly accelerated senescence. In addition, a direct interaction between WRKY53 and WRKY18 proteins could be detected in yeast using the split ubiquitin system and in planta in transiently transformed tobacco epidermal cells via FRET-FLIM. In contrast to WRKY18/18 homodimers, WRKY18/53 heterodimers positively influenced WRKY53 promoter-driven reporter gene expression but appear to act only on a shorter 1.1 kbp promoter fragment but not on a 2.8 kbp longer fragment, indicating a more complex protein-protein-DNA interaction on the longer WRKY53 promoter, most likely also triggered by the accessibility of the promoter on the chromatin level.