Golden-Gate compatible Magnaporthe oryzae Agrobacterium transformation vectors

The Golden Gate cloning system uses standardised parts to facilitate the assembly of multiple transcriptional units, to ensure that future work with these genes can be carried out with ease (Patron et al., 2015 New Phytologist, v. 208, p. 13-19).<br><div><br></div><div>We have developed the Golden Gate compatible vector pBHt2G-RFP (Addgene #107162) from the pCAMBIA-derived (Mullins et al., 2001) pBHt2G vector (Khang et al, 2010). The vector was domesticated through removal of BsaI cloning sites. An RFP-marker was inserted, which is expressed in E. coli, allowing for red-white selection of transformants. The marker is lost during the Golden Gate reaction, as it is replaced by the inserted transcriptional units.</div><div><br></div><div>Vector, sequence information and plasmid maps are available from Addgene https://www.addgene.org/107162//</div

Golden-Gate compatible Magnaporthe oryzae protoplast transformation vectors

<div>The Golden Gate cloning system uses standardised parts to facilitate the assembly of multiple transcriptional units, to ensure that future work with these genes can be carried out with ease (Patron et al., 2015 New Phytologist, v. 208, p. 13-19).</div><div><br></div><div>Three fungal transformation vectors have been adapted from the pCB1532 vector series (Sweigard et al., 1997. Fungal Genetics Newsletter 44: 52-53). Vector pCB1532B-RFP Addgene #101854 encodes bialaphos/basta/L-phosphinothricin resistance, pCB1532H-RFP #101855 hygromycin resistance and pCB1532S-RFP #101856 sulfonylurea/chlorimuron ethyl resistance. </div><div><br></div><div>Vectors were domesticated through removal of BsaI cloning sites. An RFP-marker was inserted. The RFP is expressed in E. coli, allowing for red-white selection of transformants. The marker is lost during the Golden Gate reaction, as it is replaced by the inserted transcriptional units. Vectors, sequence information and plasmid maps are available from Addgene https://www.addgene.org/plasmids/articles/28191792/ </div><div><br></div

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