40S_16_punctae2.jpg (1.19 MB)

Barley (Hordeum vulgare) ribosomal subunit 40S protein 16 (40S 16) marks chloroplasts and mitochondria when expressed in Nicotiana benthamiana.

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posted on 24.08.2017, 17:23 by Helen G Pennington, Tolga O Bozkurt, Pietro D Spanu
Barley protein 40S 16 (green) localised to chloroplasts (pink), and co-localised in pucta with an RFP-tagged mitochondrial marker (red). A) Merged colour, B) expanded RFP, C) combined RFP and GFP, and D) expanded GFP.

The 40S 16 protein was transiently expressed with a C-terminal GFP tag from vector pK7FWG2, via Agroinfiltration, along with a ribosomal marker (Geldner et al., 2009 doi: 10.1111/j.1365-313X.2009.03851.x). Leaves were harvested three days post inoculation, and transformed leaf areas mounted in water. Sample analysis was performed using a Leica SP5 resonant inverted confocal microscope with 63x objective. Excitation and emission wavelengths were 488 nm and 680 nm respectively for plastid autofluorescence, 543/588 nm for RFP and 488/495 nm for GFP. The RFP was excited with an argon laser, and GFP and autofluorescence with a helium-neon laser. Image analysis was performed using Leica LAS X and Fiji (Schindelin et al., 2012 doi 10.1038/nmeth.2019).
40S 16 accession: KP293844