Nicotiana benthamiana plastidial Methionine Sulfoxide Reductase A (NbpMSRA) localizes in the stroma of chloroplasts

The open reading frame (ORF) of NbpMSRA (SGN protein ID NbS00025360g0006.1_SGN http://solcyc.solgenomics.net/BENTH/NEW-IMAGE?type=GENE-IN-MAP&object=GJZM-26287) was amplified by PCR from N. benthamiana cDNAs. The ORF was fused with the N-terminus of a mCherry into the pICH86988 in order to create an in-frame NbpMSRA-mCherry protein fusion, under the control of a 35S-Ω promoter. The final vector was inserted into Agrobacterium tumefaciens (GV3101 strain). The NbpMSRA-mCherry protein fusion was transiently expressed in N. benthamiana leaf cells using A. tumefaciens leaf infiltration. Two days after infiltration, pavement cells were observed by live-cell imaging with a laser-scanning confocal microscope. Chlorophyll and mCherry were excited at 488 nm and 561 nm, respectively. Chlorophyll and mCherry fluorescent signals were collected between 580-620 nm and 680-700 nm, respectively. Images present a single optical section of 0.8 µm. White arrowheads indicate the NbpMSRA-mCherry signal in the stroma of the chloroplasts in the pavement cell. Scale bar: 20 µm