apoplastic side membrane marker  (Addgene inc)

Journal: bioRxiv

Article Title: An RXLR effector targets ER-Golgi interface to induce ER stress and necrotic cell death

doi: 10.1101/2023.12.15.571945

Figure Lengend Snippet: Pc12 inhibits the location of ER-resident RFP and the secretion of apoplastic membrane-anchored moxVenus, triggering ER stress separate from the effects of tunicamycin. (A) Discontinuity and puncta localization of RFP-HDEL under the expression of Pc12. GFP, GFP-Pc12 and GFP-Pc12ΔC5 were transiently expressed in transgenic N. benthamiana expressing RFP-HDEL. More than 60 z-images in 0.3 𝜇m step size were acquired by a spinning disc confocal microscope. Images were superimposed by a maximum z-projection function in ImageJ. The brightness of the original micrograms was enhanced to improve the visibility of subcellular compartments. This processing does not change the conclusion drawn from the images. RFP displayed in magenta. More than 12 images were acquired from 3 independent experiments. Scale bars, 20 𝜇m. (B) Apoplastic membrane targeted moxVenus (apoSP-moxVenus-TM) remained in ER in the existence of Pc12. RFP-Pc12, RFP-Pc12ΔC5, and RFP driven by the ethanol promoter were induced by 10% ethanol treatment. Pc12 expression inhibited the secretion of the marker proteins resulted in their accumulation in the ER. Z-images for thicker than 3 𝜇m sections in 0.75 𝜇m step size were acquired by a laser scanning confocal microscope. Images of the corresponding sections were processed to improve the brightness for the clarity. This processing does not change the conclusion drawn from the images. moxVenus was pseudo-colored to green. More than 12 images were acquired from 3 independent experiments. Scale bars, 20 𝜇m. (C) Accumulation of the ER stress marker protein (Bip) in response to Pc12 expression. Plants were treated with mock and Tm (10 ug/ml) and sampled over time. Plants expressing RFP and RFP-Pc12 after 5% ethanol treatment were sampled over time. (D) Transcripts level of UPR-related transcription factor and ER chaperon genes upon expression of Pc12. In (C), total RNA was extracted at 0 and 12 hours after tunicamycin or ethanol treatment. UPR-related genes, ER chaperon genes ( BLP4 and CRT1 ) and transcription factor genes ( bZIP28 and bZIP60 ), were normalized with NbEF1a in qRT-PCR.

Article Snippet: Plant expression constructs of Rab variants and apoplastic side membrane marker were deposited to the Addgene (Rab13-2, ID:213473; Rab 13-2 Q74L , ID:213474; Rab13-2 S29N , ID:213475; ApoSP-moxVenus-TM, ID: 213477) constructs are deposited in Addgene.

Techniques: Membrane, Expressing, Transgenic Assay, Microscopy, Marker, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Localization of planteose hydrolysis during seed germination of Orobanche minor

doi: 10.1101/2021.06.16.448768

Figure Lengend Snippet: Localization of mCherry fusion proteins transiently expressed in leaves of Nicotiana benthamiana . (A) OmAGAL2:mCherry, (E) ΔSP-OmAGAL2:mCherry, (I) SP:mCherry, and (M) mCherey were co-expressed with the apoplast marker At5g11420:pH-tdGFP (B, F, J, N). (C, D, G, H, K, L, O, P) Merged images of fluorescence from mCherry and GFP. Scale bars: 50 µm (A–C, E–G, I–K, M–O) and 20 µm (D, H, L, P).

Article Snippet: Transient expression was conducted by co-inoculation of Agrobacterium tumefaciens strain GV3101 cultures carrying each construct with those carrying the vector pMDC-At5g11420:pH-tdGFP as an apoplast marker , and pDGB3alph2_35S:P19:Tnos (GB1203, Addgene #68214) in leaves of Nicotiana benthamiana . mCherry and green fluorescent protein (GFP) were excited at 555 and 488 nm, and observed in the range of 570–600 and 490–520 nm, respectively, using a LSM700 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

Techniques: Marker, Fluorescence