Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s. (A) In vitro SUMOylation of AaMYB31s. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (B) In vivo SUMOylation of AaMYB31s in Actinidia arguta protoplasts. AaMYB31.1‐Myc or AaMYB31.2‐Myc was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies. (C) In vivo SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (D) In planta SUMOylation of AaMYB31s. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis , 35S::AaMYB31.1‐GFP , and 35S::AaMYB31.2‐GFP transgenic plants, and the AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (E) In vivo SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP in N. benthamiana leaves. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, or AaMYB31.2 K76R ‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were co‐expressed with FLAG‐SUMO1 AA as a negative control. AaMYB31.1‐GFP or AaMYB31.2‐GFP was immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (F) In planta SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP. Total proteins were extracted from freshly harvested leaves of 35S::AaMYB31.1‐GFP , 35S::AaMYB31.2‐GFP , 35S::AaMYB31.1 K76R ‐GFP , and 35S::AaMYB31.1 K76R ‐GFP transgenic plants, and the GFP‐fusion proteins were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (G) AaSIZ1 SUMOylated AaMYB31s protein in vitro. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (H) In vivo AaSIZ1 SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP and AaSIZ1 were co‐expressed with or without FLAG‐SUMO1 in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (I) The SUMOylated of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. (J) AaSIZ1 mediates the SUMOylation of AaMYB31s in Actinidia arguta protoplasts. Myc‐AaMYB31.1 or Myc‐AaMYB31.2 was co‐expressed with FLAG‐SUMO1 in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: In Vitro, In Vivo, Immunoprecipitation, Transgenic Assay, Negative Control, Incubation

Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s enhances their protein stability. (A, B) The protein degradation of AaMYB31s in the AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, and AaMYB31.2 K76R ‐GFP were detected with anti‐GFP antibody. ACTIN was used as an internal control. (C, D) In vivo protein degradation of AaMYB31s in N. benthamiana . 35S::GFP or 35S::AaSIZ1‐GFP was co‐expressed with Myc‐AaMYB31s and HA‐RFP in N. benthamiana leaves, and the total protein was extracted. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were detected with anti‐Myc antibody. HA‐RFP was used as an internal control. (E, F) The protein degradation of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. ACTIN was used as an internal control.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: Transgenic Assay, Control, In Vivo, Incubation

Journal: Virulence

Article Title: Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

doi: 10.1080/21505594.2024.2412750

Figure Lengend Snippet: Generation and verification of Hfl1p-tap (tandem affinity purification) construct. (a) Illustration outlining the integration of the Hfl1p-tap tag into the C. albicans genome. A heterozygous HFL1/hfl1∆ strain was generated using fusion PCR, employing the 5’ region of the HFL1 fragment, the 3’ region of the HFL1 fragment, and the LEU2 marker from the pSN40 plasmid. Confirmation of LEU2 prototrophic transformants (HFL-11) was achieved through PCR analysis, using the primers specified in Table S1. (b) Validation of Hfl1p-tap through Western blot analysis using an anti-TAP-Tag monoclonal antibody (1:5000). Whole-cell protein extracts from cultures grown in YPD (2% glucose) and YPG (2% glycerol) media were separated on a 10% SDS-PAGE gel. (c) Morphological resemblance of Hfl1p-tap strains (HTA6 and HTA9) to wild-type (WT) strain, evident through reduced pseudohyphae growth –the predominant growth form of the hfl1∆/hfl1∆ mutant – in YPD broth and agar medium. (d) Multiple sequence alignment of Hap5p and Hfl1p from C. albicans , and Dpb3p from S. cerevisiae (ScDpb3) was performed using EMBL-EBI’s clustal omega tool (version 1.2.4), highlighting the divergent sequence of Hfl1p from the other two proteins. Notably, the Hap4p-associated Domain#1 in Hap5p is absent in both Hfl1p and ScDpb3p.

Article Snippet: To optimize the antibody concentration for subsequent ChIP experiments, we evaluated various commercial products and found that anti-TAP-Tag mAb (ABclonal) at a 1:5000 dilution produced a clean product within the 38–52 kDa range, consistent with the 47 kDa size of TAP-tagged Hfl1p protein.

Techniques: Affinity Purification, Construct, Generated, Marker, Plasmid Preparation, Biomarker Discovery, Western Blot, SDS Page, Mutagenesis, Sequencing

Journal: Virulence

Article Title: Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

doi: 10.1080/21505594.2024.2412750

Figure Lengend Snippet: Integration of proteomic and phenotypic evidence highlighting Hfl1p’s role in carbon metabolism, mitochondrial respiration, and MDR transporters. (a) Proteomic data demonstrates reduced translation of mtDNA-encoded CI subunits (Nads), as well as nuclear-encoded CI subunits and CI regulator Goa1p, along with proteins associated with the TCA cycle in the HFL1 null mutant ( hfl1∆/hfl1∆ ). The downregulation of mitochondrial respiration and the TCA cycle is counterbalanced by elevated levels of proteins linked to non-glucose metabolism and mitochondrial transporters. (b) BN-page analysis reveals a significant decrease in the content of CI (complex I) in the hfl1∆/hfl1∆ mutant, particularly noticeable in YPD medium with 2% glucose when compared with WT and the dpb4∆/dpb∆ mutant. In YPG medium with 2% glycerol, the decrease in CI content is less evident. The quantifiable CI content from each strain, as determined by ImageJ analysis, is represented as relative ratios of CI/CIII and CI/CV intensities compared to WT [ , ]. The mean values obtained from three gel experiments underwent one-way ANOVA analysis. ”*” denotes statistical significance at p < 0.05. (c) The colocalization of Hfl1 (green) with mito-tracker red CMXRos (red). Tap-tagged Hfl1p is revealed by the primary antibody (mouse anti-TAP-Tag mAb) and secondary antibody (DyLight 488, goat anti-mouse IgG). Top row: HTA6, middle row: HTA9, bottom row: WT. Columns from left to right: DAPI (nuclei), mito-tracker red CMXRos, DyLight 488-Hfl1p, light, and merge. The orange arrows represent overlapping of Hfl1p with mitochondria, while the yellow arrows represent overlapping of Hfl1p with the nucleus.

Article Snippet: To optimize the antibody concentration for subsequent ChIP experiments, we evaluated various commercial products and found that anti-TAP-Tag mAb (ABclonal) at a 1:5000 dilution produced a clean product within the 38–52 kDa range, consistent with the 47 kDa size of TAP-tagged Hfl1p protein.

Techniques: Mutagenesis