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

<div>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.</div><div><br></div><div>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).</div><div>40S 16 accession: KP293844</div

An active form of Jasmonate Induced Protein 60 (JIP60) from Hordeum vulgare cv. Golden Promise

Jasmonate Induced protein 60 (JIP60) was amplified from barley leaves infected with Blumeria graminis (Pennington et al., 2018). A peptide N-terminal domain was excised, and replaced with either a methionine-leucine (ML) linker, or a methionine, leucine, aspartic acid & proline linker (MLDP) to produce active forms of the JIP60 protein, Figure 1. Mak et al., (2007) had previously shown that these modifications were required to produce an active maize ribosome-inactivating protein (b-32). The Gateway entry construct pDONR_JIP60mldp is available from Addgene https://www.addgene.org/104959