Journal: Journal of Virology

Article Title: The white spot syndrome virus wsv156 protein hijacks Parkin-dependent mitophagy to promote viral infection

doi: 10.1128/jvi.00418-26

Figure Lengend Snippet: Wsv156 targets mitochondria via Cq TOMM70 and triggers mito-aggresome formation. ( A ) Wsv156 expression increased following WSSV infection. Temporal expression of wsv156 at the mRNA (left panel) and protein level (right panel) was assessed by RT-qPCR and western blot, respectively, in Hpt cells after WSSV infection. ( B ) Wsv156 localized to mitochondria. Endogenous wsv156 protein in Hpt cells (left panel) and overexpressed wsv156 in HEK 293T cells (right panel) both predominantly co-fractionated with mitochondria. ( C ) Identification of host mitochondrial proteins interacting with wsv156 by affinity purification-mass spectrometry (AP-MS). ( D ) Wsv156 interacted with Cq TOMM70. Co-IP with anti-Myc beads was performed in HEK 293T cells co-transfected with wsv156-Myc and Cq TOMM70-HA. ( E ) Wsv156 expression induced mitochondrial aggregation. Confocal microscopy of HEK 293T cells showed co-localization of wsv156-GFP (green) with the mitochondrial marker Hs COX IV (red). Cells expressing GFP alone or wsv152-GFP served as controls. ( F ) Wsv156 knockdown attenuated WSSV-induced mitochondrial aggregation. Gene silencing of wsv156 in Hpt cells reduced mitochondrial clustering (red) compared to control cells at 12 hpi. NT, non-targeting dsRNA (GFP dsRNA) control; RNAi wsv156, wsv156-specific dsRNA. The right panel quantifies mitochondrial aggregation using the CI and the percentage of cells containing mito-aggresomes. Nuclei were stained with DAPI. **, P < 0.01.

Article Snippet: Co-IP was performed using anti-Myc magnetic beads (MedChemExpress, HY-K0206) as previously described ( ).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Marker, Knockdown, Control, Staining

Journal: bioRxiv

Article Title: Epistasis of two classical color genes, B and L-2 , synergistically controls carotenoid accumulation in squash

doi: 10.64898/2026.05.19.726227

Figure Lengend Snippet: CpAPRR2-A activates CpPSY-A expression and physically interacts with CpCHLH b but not CpCHLH B . A ) Y1H (yeast one-hybrid) analysis showed the binding of CpAPRR2-A (L-2) to CpPSY-A promoter (PSYpro) in vitro . Yeast cells were co-transformed with the PSYpro-pHis2.1 construct and L-2-pGADT7 plasmid, spread on DDO (double dropout medium, SD/–Leu/–Trp) and TDO (triple dropout medium, SD/–Leu/–Trp/–His with 80 mM 3-AT) for selection. Empty-vector (EV) controls show no growth. B ) Transactivation assay in N. benthamiana leaves showing L-2 activation of CpPSY-A promoter. EV and an unrelated protein CpDXS1 were used as negative controls. C ) Luciferase activity was quantified using protein extracts treated with luciferin or coelenterazine. Transactivation activity was normalized using FireflyLUC and expressed as NanoLUC/FireflyLUC with three biological replicates. Different letters above the bars indicate statistically significant differences (one-way ANOVA followed by Tukey’s multiple comparison test, P < 0.05). D ) Y2H (yeast two-hybrid) assay of CpCHLH b (b)/ CpCHLH B (B) interactions with CpAPRR2-A (L-2). B or b was fused to the N-terminal half of ubiquitin (Nub), while L-2 was fused to the C-terminal half (Cub). Yeast co-transformants were spotted in 10-fold serial dilutions onto nonselective (-LW) plate to confirm plasmid maintenance and selective plate (-LWAH) to assess interaction, followed by photograph after 3 days. E ) BiFC analysis of b or B interactions with L-2. L-2 was fused to nYFP, and b or B was fused to cYFP. L-2-nYFP/cYFP and nYFP/b-cYFP served as negative controls. Reconstituted YFP signals were detected by confocal microscopy. Scale bar = 20 µm applicable to all images. F ) Co-IP (co-immunoprecipitation) assay of b interaction with L-2. Total proteins were extracted from 3-week-old Arabidopsis leaves expressing Myc-tagged L-2 in Col-0 background or a Myc-tagged unrelated protein (negative control). Extracts were immunoprecipitated with anti-Myc beads and analyzed by immunoblotting with anti-Myc and anti-CHLH antibodies on both input and IP samples.

Article Snippet: Briefly, total proteins were extracted from leaves of Arabidopsis expressing either L-2-Myc or an unrelated protein-Myc fusion and incubated with anti-Myc magnetic beads (Miltenyi Biotec, Auburn, CA; Cat. 130-091-123).

Techniques: Expressing, Binding Assay, In Vitro, Transformation Assay, Construct, Plasmid Preparation, Selection, Transactivation Assay, Activation Assay, Luciferase, Activity Assay, Comparison, Y2H Assay, Ubiquitin Proteomics, Confocal Microscopy, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Epistasis of two classical color genes, B and L-2 , synergistically controls carotenoid accumulation in squash

doi: 10.64898/2026.05.19.726227

Figure Lengend Snippet: CpCHLH b but not CpCHLH B suppresses CpAPRR2-A-induced CpPSY-A expression. A ) Transactivation assay in Nicotiana benthamiana leaves. Agrobacterium cells carrying the CpAPRR2-A promoter (PSYpro)-LUC reporter were co-infiltrated with effectors expressing CpAPRR2-A (L-2), CpCHLH b (b), CpCHLH B (B), or the L-2+B and L-2+b combinations. Empty vector (EV) was used as a negative control. LUC signal was detected 48 h after infiltration, and representative leaf images are shown. B ) Quantification of transactivation activity using the NanoLUC/FireflyLUC dual-luciferase system. Luciferase activity was measured from protein extracts treated with luciferin for NanoLUC or coelenterazine for FireflyLUC. C ) ChIP-qPCR analysis of L-2 binding to the PSY promoter in Col-0 and gun5 backgrounds. Chromatins from L-2 and L-2 gun5 plants were immunoprecipitated with anti-Myc antibody or IgG (control). Enrichment of PSY promoter fragments was quantified by qPCR and expressed as 1.5-2% input DNA. Data in ( B ) and ( C ) are from three biological replicates and presented as mean ± SD ( n = 3). Different letters denote significant differences determined by one-way ANOVA followed by Tukey’s multiple-comparison test ( P < 0.05).

Article Snippet: Briefly, total proteins were extracted from leaves of Arabidopsis expressing either L-2-Myc or an unrelated protein-Myc fusion and incubated with anti-Myc magnetic beads (Miltenyi Biotec, Auburn, CA; Cat. 130-091-123).

Techniques: Expressing, Transactivation Assay, Plasmid Preparation, Negative Control, Activity Assay, Luciferase, ChIP-qPCR, Binding Assay, Immunoprecipitation, Control, Comparison