Journal: bioRxiv
Article Title: The N-degron pathway governs autophagy to promote thermotolerance in Arabidopsis
doi: 10.1101/2024.07.17.604022
Figure Lengend Snippet: (A) Multiple sequence alignment of UBR7 homologs in Arabidopsis , human, and mouse. Sequences were aligned using Clustal Omega online ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Conserved UBR and PHD domains are shaded in purple and blue, respectively. Consensus symbols are as follows: asterisks indicate identical residues; colons and periods indicate conserved and semiconserved substitutions, respectively. (B) R-GUS-FLAG generated through the Ub fusion technique is degraded in wild-type but stabilized in prt6-1 mutant. Ub-R/M-GUS-FLAG were expressed in Col-0 and prt6-1 protoplasts, followed by treatments with cycloheximide (100 μM) and MG132 (10 μM) for 3 h. GUS proteins were analyzed by immuniblotting with the anti-FLAG antibody, and Coomassie brilliant blue (CBB) staining served as a loading control. EV, empty vector. (C) Genomic structure of UBR7 gene. Triangle and arrows indicate the positions of the T-DNA insertion and primers used for PCR, respectively. Genomic DNA sequences are represented by exons (dark gray boxes), introns (lines), and UTRs (light gray boxes). Numbers refer to nucleotides of UBR7 gene. (D) Genotyping of ubr7 mutant. PCR using primers indicated in (C) verified T-DNA insertion. (E) RT-qPCR analysis of UBR7 expression in Col-0 and ubr7 plants. Actin2 was used as a control. Data represent means ± SD ( n = 4 biological replicates). Asterisks indicate significant differences between Col-0 and ubr7 plants ( t test; *** P < 0.001). (F) Validation of expression of GFP N -fused UBR7 (left) and UBR box (right) and GFP C -fused ATG8a, ATG8a R13A , and ATG8a 3A in transfected protoplasts. Protein lysates were prepared from Col-0 protoplasts transfected with the indicated constructs, followed by treatment with MG132 (10 μM) for 3 h. UBR7/UBR box fused with N-terminal MYC and ATG8a/ATG8a R13A /ATG8a 3A fused with C-terminal HA were used and therefore detected by immunoblotting with the anti-MYC and anti-HA antibodies, respectively. Ponceau S (PS) and Coomassie brilliant blue (CBB) staining served as loading controls. (G) BiFC assay for in vivo interaction between ATG8a and UBR box. GFP N , GFP C , and their fusions with UBR box, ATG8a, ATG8a R13A , and ATG8a 3A were co-expressed in Col-0 protoplasts, followed by treatment with MG132 (10 μM) for 3 h. Reconstituted GFP fluorescence was visualized under a confocal microscope. DAPI staining indicates the location of nuclei. UBR, UBR box; Chl, chlorophyll; BF, bright field. Bars, 10 μm.
Article Snippet: Membranes were incubated with anti-HA (Thermo Fisher Scientific, 71-5500), anti-MYC (Abcam, ab32), anti-ATG8a (Abcam, ab77003), anti-Ub (Santa Cruz, sc-8017), anti-FLAG (Abcam, ab205606), anti-mCherry (Abcam, ab183628), and anti-GFP (Santa Cruz, sc-9996) antibodies.
Techniques: Sequencing, Generated, Mutagenesis, Staining, Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Transfection, Construct, Western Blot, Bimolecular Fluorescence Complementation Assay, In Vivo, Fluorescence, Microscopy
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