A plant CitPITP1 protein-coding exon sequence serves as a promoter in bacteria

Genetic manipulation of plant genes in prokaryotes has been widely used in molecular biology, but the function of a DNA sequence is far from being fully known. Here, we discovered that a plant protein-coding gene containing the CRAL_TRIO domain serves as a promoter in bacteria. We firstly characterized CitPITP1 from Citrus, which contains the CRAL_TRIO domain, and identified a 64-bp sequence (key64) that is critical for prokaryotic promoter activity. In vitro experiments indicated that the bacterial RNA polymerase subunit RpoD specifically binds to key64. We then expanded our research to fungi, plant and animal species to identify key64-like sequences. Five such prokaryotic promoters were isolated from Amborella, Rice, Arabidopsis and Citrus. Two conserved motifs were identified, and mutation analysis indicated that the nucleotides at positions 7, 29 and 30 are crucial for key64-like transcription activity. We detected full-length recombinant CitPITP1 from E. coli, and visualized a CitPITP1-GFP fusion protein in plant cells, supporting the idea that CitPITP1 encodes a protein. However, although exon 4 of CitPITP1 contained key64, it did not demonstrate promoter activity in plants. Our study describes a new basal promoter, provides evidence for neofunction of gene elements across different kingdoms, and provides new knowledge for the modular design of promoters.