Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: Three tomato cDNAs that interact with the Pepino mosaic virus (PepMV) triple gene block protein 1 (TGBp1) identified by yeast library screening. ( A ) Amino acid (aa) multiple sequence alignment of the three identified uncharacterized interactors (p26H1, p26J1, p26D2) from a tomato cDNA library and the homologous Arabidopsis thaliana thioredoxin domain-containing protein 9 ( At TXND9; NP_179489.1). The full-length protein sequence obtained from Solanum lycopersicum ( Sl ) and the TXND9 orthologues from Solanum tuberosum ( St ; XP_006352322), Nicotiana benthamiana ( Nb ; Nbv5.1tr6261097), Nicotiana tabacum ( Nt ; XP_016486197) and Nicotiana sylvestris ( Ns ; XP_009767764) were also used. Conserved regions corresponding to the majority of the sequences are indicated with black color and with grey the different amino acids in the conserved sequences. ( B ) Interaction between PepMV TGBp1 and the isolated prey plasmids (interactors) using the yeast two-hybrid assay. Co-transformed Saccharomyces cerevisiae EGY48 cells grown in nonselective glucose/complete medium lacking histidine and tryptophan (Glu/CM-H,W) ( left ) and selective galactose-raffinose/complete medium lacking histidine, tryptophan and leucine (Gal-Raff/CM-H,W,L) ( right ) of the pairwise combinations: I, pGILDA-p26//pJG4-5/p26J1; II, pGILDA-p26//pJG4-5/p26H1; III, pGILDA-p26//pJG4-5/p26D2; IV, pGILDA//pJG4-5; V, pGILDA//pJG4-5/p26J1; VI, pGILDA/pJG4-5/p26H1; VII, pGILDA//pJG4-5/p26D2; VIII, pGILDA-p26//pJG4-5.

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Blocking Assay, Library Screening, Sequencing, cDNA Library Assay, Isolation, Y2H Assay, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: Characterization of the tomato interactor Sl TXND9. ( A ) A Neighbor-Joining tree generated using amino acid sequences of selected genes from the four protein groups that constitute the thioredoxin superfamily, the TXND9 orthologues and PLP3 sequences, was used to infer their evolutionary relationships. Accession numbers are given next to each gene. The analysis involved 50 amino acid sequences. The value on the left of each branch represents the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. ( B ) Partial amino acid sequence alignment with the tomato interactor ( Sl TXND9) identified orthologous genes from N. benthamiana ( Nb ) and A. thaliana ( At ) and selected thioredoxin genes from Sl , Nb and At that possess the WCXPC active redox motif. The asterisk indicates the tenth amino acid; W--PC vs. WCXPC motifs are highlighted, with black color the conserved W, P and C amino acids in all proteins (TXND9 orthologues and thioredoxin types proteins) and with gray color the conserved C amino acid of the thioredoxin type proteins.

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Generated, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: Pepino mosaic virus triple gene block 1 (TGBp1) interacts in vitro with Sl TXND9. Maltose-binding protein (MBP) and an MBP-TGBp1 fusion were expressed in Escherichia coli BL21 (DE3) cells along with the bacterially expressed tomato Sl TXND9 interactor labelled with the S:tag ( Sl TXND9-S:tag). MBP alone (lane 2) or MBP fused with TGBp1 (MBP-TGBp1; lane 3) was amylose affinity purified and incubated with the expressed Sl TXND9-S:tag (lanes 1–3). The eluates were analyzed for the presence of the Sl TXND9, MBP or an MBP fusion using α-S:tag ( A ) or α-MBP ( B ) antibodies. The lane M indicates the protein marker, whereas the “+” and “−“ indicate the presence or absence, respectively, of the Sl TXND9-S:tag, MBP-TGBp1 or MBP in each lane.

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Blocking Assay, In Vitro, Binding Assay, Affinity Purification, Incubation, Marker

Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: In planta subcellular localization of the Sl TXND9 interacting protein in Nicotiana benthamiana cells and bimolecular fluorescent complementation (BiFC) interaction assay. ( A ) Subcellular localization of Sl TXND9 fused with the green fluorescent protein (GFP). The fluorescence was visualized by confocal laser scanning microscopy in the cell cytoplasm. ( B ) Reconstitution of yellow fluorescent protein (YFP) fluorescence was visualized by confocal laser scanning microscopy after co-expression of the fusion proteins. ( B1 ) Co-infiltration of pCYFP- Sl TXND9 and pNYFP-TGBp1 revealing their interaction in the cell cytoplasm. ( B2 – B4 ) Co-infiltrations of pCYFP with pNYFP, pCYFP- Sl TXND9 with pNYFP, and pCYFP with pNYFP-TGBp1 (negative controls). ( B5 ) Co-infiltration of pCYFP-bZIP63 and pNYFP-bZIP63 (positive control) revealing the interaction in the nucleus. c: cytoplasm; Bars: 75 mm. The arrows in ( B1 ) indicate cell wall (c) and a nucleus (n).

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Fluorescence, Confocal Laser Scanning Microscopy, Expressing, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: Immunoblot analysis of fractionated proteins from healthy and PepMV-infected N. benthamiana plants. Examination of the compartmentalization PepMV virions and the host interactors of PepMV TGP1 and coat protein (CP), CAT1 and HSP70 respectively, and Nb TXND9 in fractionated plant extracts. Total represents the total protein extract, CW represents the cell wall fraction; P1 is the 1000× g pellet fraction; P30 is the 30,000× g pellet fraction and S30 is the 30,000× g supernatant fraction. Labels on the left indicate the antiserum used to probe each panel.

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Western Blot, Infection

Journal: International Journal of Molecular Sciences

Article Title: A Thioredoxin Domain-Containing Protein Interacts with Pepino mosaic virus Triple Gene Block Protein 1

doi: 10.3390/ijms19123747

Figure Lengend Snippet: Immuno-gold labelling (IGL) of N. benthamiana Leaf Sections with α- Sl TXND9 IgG. ( A ) Healthy tissue shows no labelling. ( B ) Cucumber mosaic virus (CMV)-infected shows no labelling. ( C – K ) PepMV infected tissue. ( C , D ) PepMV-like particles (VLP) in systemically infected leaves showing a light labelling at 4 dpi. ( E ) Labelled scattered VLPs in the vacuole of the epidermal cell of a locally-infected leaf. Note the specific labelling of the plasmodesmata (PD). ( F – K ) Specific labelling of PDs (Ch: chloroplast, m: mitochondria, ER: endoplasmic reticulum, px: peroxisome, CW: cell wall, N: nucleus; arrow in C indicates a 15 nm gold particle; bars: 500 nm).

Article Snippet: A total of 1 mg of highly pure His-TRX-S protein was used to immunize a rabbit in order to produce a polyclonal antiserum, from which specific IgG was purified using a multi-stage purification process: Total IgG was selected from the serum using a protein-A column, anti-his IgG was depleted using a his-tagged column, and Sl TXND9 IgG was selected on the basis of its cross-reactivity with MBP- Sl TXND9 (David’s Biotechnologie, Regensburg, Germany).

Techniques: Infection