Journal: Stress Biology

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

doi: 10.1007/s44154-026-00308-2

Figure Lengend Snippet: S-acylation at Cysteine-4 in C4 encoded by TYLCCxV promotes plasma membrane localization and protein expression. A Representative confocal microscopy images of subcellular localization of C4 and mutant C4 C4S in N. benthamiana leaf epidermal cells. Transgenic H2B-RFP N . benthamiana plants were infiltrated with C4-GFP or C4 C4S -GFP or GFP, respectively. H2B was used as a nucleus marker. Columns from left to right: GFP, RFP, and a GFP/RFP merged field (Merge). Scale bars, 20 μm. B Subcellular fractionation analysis of C4 and mutant C4 C4S . N. benthamiana leaves were co-infiltrated with PIP2A-DsRed and C4-GFP, C4 C4S -GFP, or GFP, respectively. Leave tissues were fractionated into soluble (S) and membrane-enriched (P) fractions after centrifugation at 3,000 g or 30,000 g (30 K). Immunoblot was used to detect anti-GFP or anti-RFP antibodies. GFP and PIP2A-DsRed acted as markers of the soluble and membrane-enriched fractions, respectively. C and E S-acylation of C4 ( C ) or mutant C4 C4S ( E ) proteins detected by the biotin-switch assay. N. benthamiana leaves were infiltrated with C4-GFP or C4 C4S -GFP, and protein extracts were loaded onto NeutraAvidin beads. Immunoblots were conducted with an anti-GFP antibody. Lanes labeled “Palmitoylation” show levels of C4-GFP or C4 C4S -GFP recovered from Neutravidin beads and indicate Palmitoylation. Hyd, hydroxylamine. D Detection of the S-acylation site in C4 via Q-exactive liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. N. benthamiana leaves were expressed with C4-Flag and protein extracts were analyzed by SDS-PAGE. The red arrow and circle point to the S-acylation site at cysteine-4. F Western blot analysis and semi-qRT-PCR assay of C4 and C4 C4S protein accumulation and mRNA levels. C4-Flag or C4 C4S -Flag was infiltrated into opposite halves of the N. benthamiana leaves, and protein extracts were detected via Western blots with an anti-Flag antibody. The amount of C4 expression was calculated using Image J 1.8.0, and the data of C4-Flag was set to 1.00. Expression of actin was used as a loading control. Transcription levels of C4 and C4 C4S were identified by semi-qRT-PCR. Actin was used as the reference gene. G and H Effects of 2-bromopalmitate (2-BP) on the subcellular localization ( G ) and protein accumulation on the transcription level of C4 ( H ). N. benthamiana leaves transiently expressing C4-GFP or C4-Flag were co-infiltrated with 500 µM 2-BP or CH 3 OH (a solution as control). Scale bars, 10 μm ( G ). Samples were detected by Western blot with an anti-Flag antibody. Protein accumulation of C4 was calculated by Image J 1.8.0, and data of C4 with CH 3 OH treatment was set to 1.00. Semi-qRT-PCR detected the transcription level of C4 mRNA ( H )

Article Snippet: Immunoblotting was performed using Mouse anti-GFP-Tag monoclonal antibody (ABclonal, Wuhan, China), Mouse anti-Flag-Tag monoclonal antibody (ABclonal), and HRP-conjugated Goat anti-Mouse or anti-Rabbit IgG antibody (ImmunoWay, Suzhou, China).

Techniques: Clinical Proteomics, Membrane, Expressing, Confocal Microscopy, Mutagenesis, Transgenic Assay, Marker, Fractionation, Centrifugation, Western Blot, Biotin Switch Assay, Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, SDS Page, Quantitative RT-PCR, Control

Journal: Stress Biology

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

doi: 10.1007/s44154-026-00308-2

Figure Lengend Snippet: Interaction of C4 with NbPAT4 in vitro and in vivo. A Yeast Split-ubiquitin assay of the interaction between TYLCCxV C4 and four palmitoyl acyltransferases (NbPAT1-2, NbPAT4, and NbPAT6). The yeast strain NMY51 co-transformed with the indicated plasmids was subjected to tenfold serial dilutions and grown on a SD/-Trp/-Leu/-His/-Ade medium. Strains carried combinations of pPR3-N-p53/pDHB1-largeT were used as positive control, and those with empty pPR3-N/PDHB1-largeT were used as negative control. B BiFC analysis showing the interaction of C4 and NbPAT4. The transgenic H2B-RFP N . benthamiana plants were co-infiltrated with 2YN-C4 and 2YC-NbPAT4, 2YN-C4, and 2YC, or 2YN and 2YC-NbPAT4, respectively. H2B was used as a nucleus marker. Columns from top to bottom: YFP, RFP, and Merge. Scale bars, 20 μm. C Interaction between TYLCCxV C4 and NbPAT4 via the Co-IP assay. N. benthamiana leaves co-infiltrated the NbPAT4-Flag with C4-GFP or GFP. Total protein extracts were incubated with anti-GFP mAb-magnetic beads. The input and the co-immunoprecipitated proteins were detected by Western blot with an anti-GFP or anti-Flag antibody

Article Snippet: Immunoblotting was performed using Mouse anti-GFP-Tag monoclonal antibody (ABclonal, Wuhan, China), Mouse anti-Flag-Tag monoclonal antibody (ABclonal), and HRP-conjugated Goat anti-Mouse or anti-Rabbit IgG antibody (ImmunoWay, Suzhou, China).

Techniques: In Vitro, In Vivo, Split-ubiquitin Assay, Transformation Assay, Positive Control, Negative Control, Transgenic Assay, Marker, Co-Immunoprecipitation Assay, Incubation, Magnetic Beads, Immunoprecipitation, Western Blot

Journal: Stress Biology

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

doi: 10.1007/s44154-026-00308-2

Figure Lengend Snippet: NbPAT4 modulates TYLCCxV infection and participates in S-palmitoylation of C4. A and C Symptoms induced by TYLCCxV in wild type (WT) and transgenic Nbpat4 ( A ) or OE-NbPAT4 N. benthamiana ( C ) at 15/30 dpi. Scale bars, 2 cm. B and D Southern blot analysis of TYLCCxV DNA accumulation in N. benthamiana plants shown in panel a ( B ) or panel c ( D ) at 15/30 dpi. Total genomic DNA (10 μg per lane) from a mixture of three seedlings was used in Southern blot. The blot was probed with the CP of TYLCCxV. The position of the ssDNA form is indicated. E S-acylation levels of C4 protein detected by the biotin-switch assay in wild type N. benthamiana (WT), the transgenic Nbpat4 , or OE-NbPAT4 plants. Samples were loaded onto NeutraAvidin beads and detected via an immunoblot with the anti-GFP antibody. Lanes labeled “Palmitoylation” show levels of C4-GFP recovered from NeutrAvidin beads and indicate Palmitoylation. Hyd, hydroxylamine. The S-acylation level and protein accumulation of C4 are calculated using Image J 1.8.0, and the data of C4 with Hyd treatment in wild type plants was set to 1.00. F S-acylation levels of C4 shown in panel e quantified using GraphPad Prism 8. The S-acylation level of C4 in wild type N. benthamiana plants was set to 1.0. Data are presented as means and SD of three biological replicates. Two asterisks mark significant differences between two treatments at p < 0.01via Student’s t -test

Article Snippet: Immunoblotting was performed using Mouse anti-GFP-Tag monoclonal antibody (ABclonal, Wuhan, China), Mouse anti-Flag-Tag monoclonal antibody (ABclonal), and HRP-conjugated Goat anti-Mouse or anti-Rabbit IgG antibody (ImmunoWay, Suzhou, China).

Techniques: Infection, Transgenic Assay, Southern Blot, Biotin Switch Assay, Western Blot, Labeling

Journal: Stress Biology

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

doi: 10.1007/s44154-026-00308-2

Figure Lengend Snippet: Interaction of C4 with NbABHD6 in vitro and in vivo and a schematic diagram of NbABHD6. A Screening of de-S-acylation enzymes interacting with TYLCCxV C4 by the Yeast Split-ubiquitin assay. Strains carried combinations of pPR3-N-p53/pDHB1-largeT and the empty pPR3-N/pDHB1-C4 or largeT were used as positive and negative controls, respectively. B Yeast Split-ubiquitin assay of the interaction with TYLCCxV C4 and NbABHD6. C Interaction of TYLCCxV C4 and NbABHD6 via the BiFC assay. Transgenic H2B-RFP N . benthamiana plants were co-infiltrated with 2YN-C4 and 2YC-NbABHD6, 2YN-C4 and 2YC, or 2YN and 2YC-NbABHD6, respectively. Scale bars, 20 μm. D Co-IP assay showing the interaction of TYLCCxV C4 and NbABHD6. N. benthamiana leaves were co-infiltrated C4-Flag with NbABHD6-GFP or GFP, and protein extracts are incubated with anti-GFP mAb-magnetic beads. The input and co-immunopreciptated proteins are detected by immunoblot with an anti-GFP or anti-Flag antibody. E Schematic diagram of the NbABHD6 protein structure. ABHD: αlpha/beta hydrolase domain. Numbers represent the amino acid positions of the NbABHD6 protein. Three residues (S147, D212, and H241) involved in catalysis of NbABHD6 are indicated by the black spots. S147, serine at 147; D212, aspartate at 212; H241, histidine at 241

Article Snippet: Immunoblotting was performed using Mouse anti-GFP-Tag monoclonal antibody (ABclonal, Wuhan, China), Mouse anti-Flag-Tag monoclonal antibody (ABclonal), and HRP-conjugated Goat anti-Mouse or anti-Rabbit IgG antibody (ImmunoWay, Suzhou, China).

Techniques: In Vitro, In Vivo, Split-ubiquitin Assay, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Co-Immunoprecipitation Assay, Incubation, Magnetic Beads, Western Blot

Journal: Stress Biology

Article Title: Reversible S-palmitoylation of C4 protein encoded by TYLCCxV orchestrates geminiviral pathogenesis

doi: 10.1007/s44154-026-00308-2

Figure Lengend Snippet: NbABHD6 enhances degradation of TYLCCxV C4 protein by the 26S proteasome pathway. A Western blot analysis and semi-qRT-PCR assay of the TYLCCxV C4 protein and mRNA levels in C4-Flag with NbABHD6-GFP or GFP co-infiltrated N. benthamiana leaves. Samples were detected via immunoblot with anti-Flag or anti-GFP antibodies. The protein accumulation of C4-Flag was calculated by Image J 1.8.0, and the data of C4 co-infiltrated with GFP were set as 1.00. Ponceau red stained Rubisco was used as a loading control. Transcription levels of C4 were identified by semi-qRT-PCR. Actin was used as the reference gene. B Analysis of C4 protein accumulation shown in panel a using GraphPad Prism 8. The data in the C4-Flag and GFP treatments were set to 1.0. Data are presented as means and SD of three biological replicates. Two asterisks mark differences between two treatments at p < 0.01 via Student’s t -test. C and D S-acylation levels ( C ) and protein accumulation levels ( D ) of the C4 protein detected by the biotin switch assay and Western blot analysis. N. benthamiana was co-infiltrated with C4-Flag and GFP, NbABHD6 (mSDH)-GFP, or NbABHD6-GFP, respectively. Protein extracts were analyzed by Western blot with anti-Flag or anti-GFP antibodies. S-acylation and protein accumulation levels of C4 were calculated using Image J 1.8.0, and the data of C4 co-expressed with GFP was set to 1.00. Lanes labeled “Palmitoylation” show the levels of C4-Flag recovered from the NeutrAvidin beads and indicate Palmitoylation. E Interaction of NbABHD6 (mSDH) and C4 by the Yeast Split-ubiquitin assay. F Semi-in vivo degradation inhibition analysis of C4 using the protease inhibitors MG132, E64d, or 3MA. The N. benthamiana leaves transiently expressing C4-Flag were treated with 100 μM CHX together with 100 μM MG132, 100 μM E64d, 5 mM E64d, or DMSO. The relative accumulation level of C4 was calculated using Image J 1.8.0, and the data of C4 in DMSO treatments was set as 1.00. G Semi-in vivo C4 protein stability assay by Western blot. N. benthamiana leaves transiently co-expressed C4-Flag with NbABHD6-GFP or GFP were treated with 100 μM MG132 or DMSO, respectively. The amount of C4 expressing was calculated using Image J 1.8.0, and the data of C4 co-infiltrated with GFP in DMSO-treated leaves was set as 1.00. H Protein accumulation of C4 analyzed by Western blot. N. benthamiana co-expressed with C4-Flag and GFP, NbABHD6 (mSDH)-GFP, or NbABHD6-GFP were treated with 100 μM MG132. The accumulation level of C4 was calculated using Image J 1.8.0, and the data of C4 co-infiltrated with GFP was set as 1.00

Article Snippet: Immunoblotting was performed using Mouse anti-GFP-Tag monoclonal antibody (ABclonal, Wuhan, China), Mouse anti-Flag-Tag monoclonal antibody (ABclonal), and HRP-conjugated Goat anti-Mouse or anti-Rabbit IgG antibody (ImmunoWay, Suzhou, China).

Techniques: Western Blot, Quantitative RT-PCR, Staining, Control, Biotin Switch Assay, Labeling, Split-ubiquitin Assay, In Vivo, Inhibition, Expressing, Stability Assay