Identification of NMD regulated genes in Nicotiana benthamiana

Nonsense mediated decay (NMD) is a translation termination coupled quality control system. NMD identifies and degrades aberrant mRNAs containing premature termination codons, thereby preventing the accumulation of detrimental truncated proteins. NMD also controls the expression of several normal mRNAs and non-coding transcripts. RNA-seq assays were conducted to identify the NMD regulated genes in Arabidopsis. However, NMD targets have not been studied in other plants. To identify the conserved NMD targets, we wanted to identify the NMD regulated genes in Nicotiana benthamiana model species. Virus induced gene silencing was used to inactivate UPF1 NMD key factor in N. benthamiana, and then comparative RNA-seq experiment was carried out using UPF1-silenced and control–silenced plants. We found that in N. benthamiana significantly more genes are controlled by NMD than in Arabidopsis and that, surprisingly few conserved NMD targets can be recognized. These results suggest that while the mechanism of NMD is highly conserved, the set of NMD controlled genes can be quickly changed in plants. Comparative Illumina RNA-seq experiments were conducted from leaf samples isolated from three UPF1-silenced and three control silenced plants. Virus induced gene silencing was used to generate the silenced plants. In the control lines, only the phytoene desaturase (PDS) silencing monitor gene was inactivated, while in the UPF1-silenced plants, both PDS and UPF1 genes were silenced. Sequencing libraries were prepared from polyA enriched RNA samples.