DRP004402
Genome-wide analysis of transcription start sites and promoter motifs of phytoplasma
Phytoplasmas are obligate intracellular parasitic bacteria of both plants and insects. We previously identified the RpoD-dependent consensus promoter sequence of phytoplasma using an in vitro transcription assay. However, genome-wide landscape of RNA transcripts (including non-coding RNA) and RpoD-independent promoter elements were still unknown. In this study, we performed an improved RNA sequencing (RNA-seq) analysis for the genome-wide identification of the transcription start sites (TSSs) and the consensus promoter sequences. We constructed cDNA libraries using a random adenine/thymine hexamer primer in addition to a conventional random hexamer primer for efficient sequencing of 5'' termini of phytoplasmal RNAs. We identified 231 TSSs and they were classified into four categories: mRNA TSSs (mTSSs), internal sense TSSs (iTSSs), antisense TSSs (asTSSs) and orphan TSSs (oTSSs). The average distance from translation start codons to mTSSs was similar to that of mycoplasma. We found almost iTSSs possessed alternative start codons located downstream of them. Using the information of the asTSSs and oTSSs, we found evidence of new open reading frames. The presence of asTSSs and oTSSs indicated the transcription of antisense RNAs, which might act as non-coding regulatory RNAs in phytoplasmas. Using a de novo motif discovery program, we identified two consensus motif sequences located upstream of the TSSs. One of the motifs was almost identical to the RpoD-dependent consensus promoter sequence, while the other one was an unidentified novel motif. We identified a novel consensus motif sequence located upstream of TSSs. The novel consensus motif sequence may be recognized by FliA, an alternative transcription initiation factor of phytoplasmas. We also detected genome-wide phytoplasmal non-coding RNAs for the first time, which may regulate phytoplasmal gene expression as cis- and/or trans-regulatory elements. These findings will be valuable in understanding the regulation mechanism of phytoplasmal gene expression.