scholarly journals Silencing Suppressor Protein VPg of a Potyvirus Interacts With the Plant Silencing-Related Protein SGS3

2014 ◽  
Vol 27 (11) ◽  
pp. 1199-1210 ◽  
Author(s):  
Minna-Liisa Rajamäki ◽  
Janne Streng ◽  
Jari P. T. Valkonen
Keyword(s):  
Rna Silencing ◽  
Bimolecular Fluorescence Complementation ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Main Determinant ◽  
Potato Virus A ◽  
Cytoplasmic Bodies ◽  
Multiple Protein ◽  
Suppressor Of Rna Silencing ◽  
Two Hybrid

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg–SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg–SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

scholarly journals Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system

2001 ◽  
Vol 82 (4) ◽  
pp. 935-939 ◽  
Author(s):  
Deyin Guo ◽  
Minna-Liisa Rajamäki ◽  
Mart Saarma ◽  
Jari P. T. Valkonen
Keyword(s):  
Hybrid System ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Yeast Two Hybrid ◽  
Potato Virus A ◽  
Yeast Two Hybrid System ◽  
Multiple Protein ◽  
Interaction Map ◽  
Two Hybrid ◽  
Shed Light

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus Potyvirus, family Potyviridae) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC–HC, HC–CI, VPg–VPg, NIa–NIb and CP–CP) and weak but reproducible interactions were detected for seven additional combinations (P1–CI, P3–NIb, NIaPro–NIb, VPg–NIa, VPg–NIaPro, NIaPro–NIa and NIa–NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC–HC, VPg–VPg, VPg–NIa, NIa–NIa and NIa–NIb) and weaker but reproducible interactions were detected for three additional combinations (P3–NIa, NIa–NIaPro and CP–CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg–NIa, NIa–NIaPro and NIa–NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

scholarly journals The role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

2021 ◽  
Author(s):  
Jinxin Gao ◽  
Jie Chen
Keyword(s):  
Toxin Production ◽  
Bimolecular Fluorescence Complementation ◽  
Curvularia Lunata ◽  
Yeast Two Hybrid ◽  
Acetyl Xylan Esterase ◽  
Btb Domain ◽  
Intermediate Metabolites ◽  
Regulative Mechanism ◽  
Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular fluorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

scholarly journals Identification of Beet necrotic yellow vein virus P25 Pathogenicity Factor–Interacting Sugar Beet Proteins That Represent Putative Virus Targets or Components of Plant Resistance

2009 ◽  
Vol 22 (8) ◽  
pp. 999-1010 ◽  
Author(s):  
Heike Thiel ◽  
Mark Varrelmann
Keyword(s):  
Sugar Beet ◽  
Resistance Mechanism ◽  
Yellow Vein ◽  
Bimolecular Fluorescence Complementation ◽  
Plant Proteins ◽  
Yeast Two Hybrid ◽  
Response To Stress ◽  
Bimolecular Fluorescence Complementation Assay ◽  
Two Hybrid ◽  
Important Disease

Beet necrotic yellow vein virus (BNYVV) induces the most important disease threatening sugar beet. The growth of partially resistant hybrids carrying monogenic dominant resistance genes stabilize yield but are unable to entirely prevent virus infection and replication. P25 is responsible for symptom development and previous studies have shown that recently occurring resistance-breaking isolates possess increased P25 variability. To better understand the viral pathogenicity factor's interplay with plant proteins and to possibly unravel the molecular basis of sugar beet antivirus resistance, P25 was applied in a yeast two-hybrid screen of a resistant sugar beet cDNA library. This screen identified candidate proteins recognized as orthologues from other plant species which are known to be expressed following pathogen infection and involved in plant defense response. Most of the candidates potentially related to host-pathogen interactions were involved in the ubiquitylation process and plants response to stress, and were part of cell and metabolism components. The interaction of several candidate genes with P25 was confirmed in Nicotiana benthamiana leaf cells by transient agrobacterium-mediated expression applying bimolecular fluorescence complementation assay. The putative functions of several of the candidates identified support previous findings and present first targets for understanding the BNYVV pathogenicity and antivirus resistance mechanism.

scholarly journals Translocation of Tomato Bushy Stunt Virus P19 Protein into the Nucleus by ALY Proteins Compromises Its Silencing Suppressor Activity

2006 ◽  
Vol 80 (18) ◽  
pp. 9064-9072 ◽  
Author(s):  
Tomas Canto ◽  
Joachim F. Uhrig ◽  
Maud Swanson ◽  
Kathryn M. Wright ◽  
Stuart A. MacFarlane
Keyword(s):  
Rna Silencing ◽  
Tomato Bushy Stunt Virus ◽  
Bimolecular Fluorescence Complementation ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Long Distance ◽  
Suppressor Activity ◽  
Recognition Motif ◽  
Silencing Suppression ◽  
Suppressor Of Rna Silencing

ABSTRACT The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.

Key elements of the RNAi pathway are regulated by hepatitis B virus replication and HBx acts as a viral suppressor of RNA silencing

2014 ◽  
Vol 462 (2) ◽  
pp. 347-358 ◽  
Author(s):  
Mahendran Chinnappan ◽  
Avishek Kumar Singh ◽  
Pavan Kumar Kakumani ◽  
Gautam Kumar ◽  
Sheetalnath Babasaheb Rooge ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Rna Silencing ◽  
Rnai Pathway ◽  
Mrna Levels ◽  
Silencing Suppressor ◽  
Chronic Hepatitis B Infection ◽  
Hbv Replication ◽  
B Virus ◽  
Protein Functions ◽  
Suppressor Of Rna Silencing

HBV replication is associated with reduced Drosha, Dicer and Ago2 mRNA levels in the human clinical condition of chronic hepatitis B infection. HBx protein functions as RNA-silencing suppressor by blocking the Dicer-mediated siRNA generation.

Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1173-1173
Author(s):  
Laurens T. van der Meer ◽  
Jurgen A.F. Marteijn ◽  
Theo M. de Witte ◽  
Joop H. Jansen ◽  
Bert A. van der Reijden
Keyword(s):  
Ubiquitin Ligase ◽  
Half Life ◽  
Proteasome Inhibitors ◽  
Ring Finger ◽  
Ubiquitin Ligases ◽  
Proteasomal Degradation ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Protein Levels ◽  
Two Hybrid

Abstract The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover. Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.

scholarly journals Regulation of Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Expression by F-Box/LRR-Repeat (FBXL) Protein in Wheat (Triticum aestivum L.)

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1606
Author(s):  
Min Jeong Hong ◽  
Jin-Baek Kim ◽  
Yong Weon Seo ◽  
Dae Yeon Kim
Keyword(s):  
Triticum Aestivum ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Triticum Aestivum L ◽  
26S Proteasome ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Scf Complex ◽  
Two Hybrid

F-box proteins are substrate recognition components of the Skp1-Cullin-F-box (SCF) complex, which performs many important biological functions including the degradation of numerous proteins via the ubiquitin–26S proteasome system. In this study, we isolated the gene encoding the F-box/LRR-repeat (FBXL) protein from wheat (Triticum aestivum L.) seedlings and validated that the TaFBXL protein is a component of the SCF complex. Yeast two-hybrid assays revealed that TaFBXL interacts with the wheat glycosylphosphatidylinositol-anchored protein (TaGPI-AP). The green fluorescent protein (GFP) fusion protein of TaFBXL was detected in the nucleus and plasma membrane, whereas that of TaGPI-AP was observed in the cytosol and probably also plasma membrane. yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed that TaFBXL specifically interacts with TaGPI-AP in the nucleus and plasma membrane, and TaGPI-AP is targeted by TaFBXL for degradation via the 26S proteasome system. In addition, TaFBXL and TaGPI-AP showed antagonistic expression patterns upon treatment with indole-3-acetic acid (IAA), and the level of TaGPI-AP was higher in tobacco leaves treated with both MG132 (proteasome inhibitor) and IAA than in leaves treated with either MG132 or IAA. Taken together, our data suggest that TaFBXL regulates the TaGPI-AP protein level in response to exogenous auxin application.

scholarly journals Key role of the motor protein Kinesin 13B in the activity of homeodomain-leucine zipper I transcription factors

2020 ◽  
Vol 71 (20) ◽  
pp. 6282-6296
Author(s):  
Virginia Natali Miguel ◽  
Karina Fabiana Ribichich ◽  
Jorge Ignacio Giacomelli ◽  
Raquel Lia Chan
Keyword(s):  
Transcription Factors ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Vascular Tissue ◽  
Expression Patterns ◽  
Motor Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Cauline Leaf ◽  
Two Hybrid

Abstract The sunflower (Helianthus annuus) homeodomain-leucine zipper I transcription factor HaHB11 conferred differential phenotypic features when it was expressed in Arabidopsis, alfalfa, and maize plants. Such differences were increased biomass, seed yield, and tolerance to flooding. To elucidate the molecular mechanisms leading to such traits and identify HaHB11-interacting proteins, a yeast two-hybrid screening of an Arabidopsis cDNA library was carried out using HaHB11 as bait. The sole protein identified with high confidence as interacting with HaHB11 was Kinesin 13B. The interaction was confirmed by bimolecular fluorescence complementation and by yeast two-hybrid assay. Kinesin 13B also interacted with AtHB7, the Arabidopsis closest ortholog of HaHB11. Histochemical analyses revealed an overlap between the expression patterns of the three genes in hypocotyls, apical meristems, young leaves, vascular tissue, axillary buds, cauline leaves, and cauline leaf nodes at different developmental stages. AtKinesin 13B mutants did not exhibit a differential phenotype when compared with controls; however, both HaHB11 and AtHB7 overexpressor plants lost, partially or totally, their differential phenotypic characteristics when crossed with such mutants. Altogether, the results indicated that Kinesin 13B is essential for the homeodomain-leucine zipper transcription factors I to exert their functions, probably via regulation of the intracellular distribution of these transcription factors by the motor protein.

scholarly journals Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants

2005 ◽  
Vol 126 (4) ◽  
pp. 325-337 ◽  
Author(s):  
Geneviève M. Brunet ◽  
Edith Gagnon ◽  
Charles F. Simard ◽  
Nikolas D. Daigle ◽  
Luc Caron ◽  
...  
Keyword(s):  
Structural Organization ◽  
Apical Membrane ◽  
Splice Variants ◽  
Mrna Levels ◽  
Hybrid Mapping ◽  
Yeast Two Hybrid ◽  
Additional Insight ◽  
Operational Characteristics ◽  
Mapping Analysis ◽  
Two Hybrid

The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.

scholarly journals The Peptidyl Prolyl cis/trans Isomerase FKBP38 Determines Hypoxia-Inducible Transcription Factor Prolyl-4-Hydroxylase PHD2 Protein Stability

2007 ◽  
Vol 27 (10) ◽  
pp. 3758-3768 ◽  
Author(s):  
Sandra Barth ◽  
Jutta Nesper ◽  
Philippe A. Hasgall ◽  
Renato Wirthner ◽  
Katarzyna J. Nytko ◽  
...  
Keyword(s):  
Protein Stability ◽  
Transcriptional Activity ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Isomerase Activity ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Α Subunits ◽  
Two Hybrid

ABSTRACT The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

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