Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A–C) In vitro auto-ubiquitination assays using (A) MBP-PUB32 FL compared to catalytic mutants MBP-PUB32 FL-W768A and MBP-PUB32 ΔUbox ; (B) His 6 -MBP-PUB33 FL compared to His 6 -MBP-PUB33 FL-W796A and His 6 -MBP-PUB33 ΔUbox ; (C) MBP-PUB50 FL compared to MBP-PUB50 ΔUbox . Reaction mixtures included ubiquitin, ubiquitin activating protein 1 (UBA1) and ubiquitin conjugating protein 8 (UBC8). Western blots were probed with antibodies against ubiquitin (anti-Ub) or maltose binding protein (anti-MBP); protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). (D-E) In vitro auto-phosphorylation assays of His 6 -MBP-PUB33 FL , His 6 -MBP-PUB33 KD , MBP-PUB32 FL , MBP-PUB32 KD , MBP-PUB50 FL , MBP-PUB50 KD , and His 6 -MBP-PUB51 FL ( D ) and His 6 -MBP-PUB33 FL , MBP-PUB33 FL-D603A , His 6 -MBP-PUB33 KD , and MBP-PUB33 KD-D603A ( E ). Autoradiographs (Autorad) show incorporation of γ-³²P, and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). Asterisks indicate the respective proteins. All assays were repeated at least three times with similar results by TD.

Article Snippet: The following antibodies were used, as indicated in the figure captions: anti-Ub (Cell Signaling Technology; P4D1); anti-MBP (New England Biolabs; E8032S); anti-HA (Roche; 12013819001); anti-Myc (Cell Signaling Technology; 9B11); anti-GFP (Roche; 1814460001 or ChromoTek; 3h9-150); anti-FLAG-HRP (Sigma; A8592); anti-mouse (Sigma, A0168); anti-mouse (LiCOR Biosciences; IRDye 800CW); anti-rat (Sigma; A5795).

Techniques: In Vitro, Ubiquitin Proteomics, Western Blot, Binding Assay, Staining, Phospho-proteomics

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) In vitro auto-ubiquitination assays comparing His 6 -MBP-PUB33 FL with MBP-PUB33 FL-D603A . The reaction mixture included ubiquitin, ubiquitin activating protein 1 (UBA1) or ubiquitin conjugating protein 8 (UBC8). Western blots were probed with antibodies against ubiquitin (anti-Ub) and maltose-binding protein (anti-MBP) and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). These assays were repeated more than three times with similar results by TD. (B) PUB33 phosphopeptides detected following in vitro His 6 -MBP-PUB33 FL autophosphorylation. Each peptide was identified in at least 2/3 independent replicates and was absent in control samples without ATP. Phosphosites are indicated in bold, with positions shown on the right. The schematic below indicates their location on PUB33 FL ; colours indicate the domains of PUB33 as defined in . Kinase assays were performed by TD; trypsin digestion and LC-MS/MS analysis were performed by MCRG. (C) Multiple sequence alignment of the regions including the PUB33 phosphosites across the PUB-VI/RLCK-IXb proteins. Analysis by TD. (D-E) In vitro auto-ubiquitination assays comparing His 6 -MBP-PUB33 FL with the indicated variants. Reaction mixtures include UBA1 and UBC8, with (+) and without (-) ubiquitin for the indicated times. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining with CBB. These assays were repeated more than three times with similar results by TD. (F) In vitro auto-phosphorylation assays comparing His 6 -MBP-PUB33 FL to PUB33 FL-T333A and PUB33 FL-T333D . Autoradiography (Autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB. These assays were repeated more than three times with similar results by TD. (G) Alignment of the predicted coiled-coil domain of the PUB33 homomer in its unphosphorylated (magenta) and phosphorylated at T333 (grey) configurations, as predicted by AlphaFold3. Although we noticed a modest shift in this region, the predicted PUB33 and PUB33-pT333 homomers are excellently aligned overall, with a sequence alignment score of 2264.2 and a root mean square deviation of 0.713 Å. Analysis by JM. (H) Yeast 2-hybrid between the indicated PUB33 variants. EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. Assays conducted by IK at least three times with similar results.

Article Snippet: The following antibodies were used, as indicated in the figure captions: anti-Ub (Cell Signaling Technology; P4D1); anti-MBP (New England Biolabs; E8032S); anti-HA (Roche; 12013819001); anti-Myc (Cell Signaling Technology; 9B11); anti-GFP (Roche; 1814460001 or ChromoTek; 3h9-150); anti-FLAG-HRP (Sigma; A8592); anti-mouse (Sigma, A0168); anti-mouse (LiCOR Biosciences; IRDye 800CW); anti-rat (Sigma; A5795).

Techniques: In Vitro, Ubiquitin Proteomics, Western Blot, Binding Assay, Staining, Control, Liquid Chromatography with Mass Spectroscopy, Sequencing, Phospho-proteomics, Autoradiography, Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) Unique PUB32 peptides identified by mass spectrometry following immunoprecipitation of PUB33-GFP from Col-0/35S:PUB33-GFP line #9-4. Peptides were present in 3/3 PUB33-GFP samples and 0/3 Col-0 samples; see Supplementary Table S2 for more details. Immunoprecipitation performed by TD; sample processing and analysis performed by MCRG. (B,F) Yeast 2-hybrid between PUB32 FL and PUB33 FL (B) and PUB33 FL variants (F) . EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. DB indicates constructs translationally fused to the Locus for X-Ray Sensitivity A (LexA) DNA binding domain; AD indicates constructs translationally fused to the B42 transcriptional activation domain. Assays conducted by IK at least three times with similar results. (C) Possible configuration of a PUB32:PUB33 homomer, as predicted by AlphaFold2. The colours match the labels in A. Average predicted Local Distance Difference Test (pLDDT) score for the PUB32 monomer is 75.31 and the pLDDT score for the PUB33 monomer is 77.9. The predicted template modeling (pTM) and interface pTM (ipTM) scores for the PUB32:PUB33 heteromer are 0.50 and 0.49, indicating low-to-moderate confidence in both the overall model and the predicted interface. Analysis by TD. (D) In vitro auto-ubiquitination assays comparing 2 μg His 6 -MBP-PUB33 FL in the presence of increasing amounts of MBP-PUB32 FL . Reaction mixtures include UBA1, UBC8, and ubiquitin. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB) R250. These assays were repeated more than three times with similar results by TD. (E) In vitro auto-phosphorylation assays comparing 2 μg His 6 -MBP-PUB33 FL in the presence of increasing amounts of MBP-PUB32 FL . Autoradiography (Autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB G250. Assays conducted at least three times by TD with similar results.

Article Snippet: The following antibodies were used, as indicated in the figure captions: anti-Ub (Cell Signaling Technology; P4D1); anti-MBP (New England Biolabs; E8032S); anti-HA (Roche; 12013819001); anti-Myc (Cell Signaling Technology; 9B11); anti-GFP (Roche; 1814460001 or ChromoTek; 3h9-150); anti-FLAG-HRP (Sigma; A8592); anti-mouse (Sigma, A0168); anti-mouse (LiCOR Biosciences; IRDye 800CW); anti-rat (Sigma; A5795).

Techniques: Mass Spectrometry, Immunoprecipitation, Plasmid Preparation, Control, Transformation Assay, Construct, Binding Assay, Activation Assay, In Vitro, Ubiquitin Proteomics, Western Blot, Staining, Phospho-proteomics, Autoradiography

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) Yeast 2-hybrid between the N-terminal domain of RipV1 (RipV1 NT ) and PUB32 FL , PUB33 FL , and the indicated PUB33 variants. EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. DB indicates constructs translationally fused to the Locus for X-Ray Sensitivity A (LexA) DNA binding domain; AD indicates constructs translationally fused to the B42 transcriptional activation domain. Assays conducted by JC and IK at least three times with similar results. (B-D) In vitro trans-ubiquitination of PUB33 FL-W796A (B) and PUB32 FL by RipV1 FL (C) ; or RipV1 C452A by PUB33 FL (D) . Reaction mixtures include UBA1, UBC8, and ubiquitin. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining Coomassie Brilliant Blue (CBB) R250. These assays were repeated more than three times with similar results by TD. (E) In vitro trans-phosphorylation assay between PUB33 FL or PUB33 KD and RipV1, compared to the kinase-dead variants containing D603A. Autoradiography (autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB G250. Asterisks indicate proteins of interest. Assays were repeated 3 times by TD. (F) Cell death induced by transient expression of RipV1 FL -FLAG in the presence or absence of PUB33-HA or free GFP in N. benthamiana . False color photographs were taken at 3 days post infiltration (dpi); the circles indicate the infiltrated area. Dark areas indicate cell death. Quantum yield of chlorophyll (QY) measurements were also taken at 3 dpi and are summarized in the lower histogram. Values are quantum yield (Fv/Fm) from 3 independent experiments (n=9 leaf discs per experiment). The midline represents the median; interquartile ranges are represented by the boxes, and maximum and minimum values are represented by the whiskers. Significantly different groups are labelled with lower-case letters, based on a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (p<0.0001). These assays were performed by JC and TD more than three times each in independent labs, with similar results. (G) Samples from the experiments shown in G were taken at 35 hours post infiltration (hpi) and subjected to western blot. Proteins are detected by anti-FLAG and anti-GFP antibodies; protein loading is indicated by Ponceau S staining. These assays were performed by JC three times with similar results.

Article Snippet: The following antibodies were used, as indicated in the figure captions: anti-Ub (Cell Signaling Technology; P4D1); anti-MBP (New England Biolabs; E8032S); anti-HA (Roche; 12013819001); anti-Myc (Cell Signaling Technology; 9B11); anti-GFP (Roche; 1814460001 or ChromoTek; 3h9-150); anti-FLAG-HRP (Sigma; A8592); anti-mouse (Sigma, A0168); anti-mouse (LiCOR Biosciences; IRDye 800CW); anti-rat (Sigma; A5795).

Techniques: Plasmid Preparation, Control, Transformation Assay, Construct, Binding Assay, Activation Assay, In Vitro, Ubiquitin Proteomics, Western Blot, Staining, Phospho-proteomics, Autoradiography, Expressing