scholarly journals Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3

2022 ◽  
Author(s):  
Tongtong Jin ◽  
Jinlong Yin ◽  
Song Xue ◽  
Bowen Li ◽  
Tingxuan Zong ◽  
...  
Keyword(s):  
Soybean Mosaic Virus ◽  
Direct Interaction ◽  
Chromosome 2 ◽  
Internal Deletion ◽  
Yeast Two Hybrid ◽  
Viral Pathogens ◽  
Virulence Determinant ◽  
Glycine Max L ◽  
Two Hybrid ◽  
Avirulent Isolate

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 × susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle vi-rus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA fragement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3.

scholarly journals Interaction Study of Soybean mosaic virus Proteins with Soybean Proteins using the Yeast-Two Hybrid System

2007 ◽  
Vol 23 (4) ◽  
pp. 281-286 ◽  
Author(s):  
Jang-Kyun Seo ◽  
Sung-Hyun Hwang ◽  
Sung-Hwan Kang ◽  
Hong-Soo Choi ◽  
Su-Heon Lee ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Hybrid System ◽  
Soybean Mosaic Virus ◽  
Interaction Study ◽  
Yeast Two Hybrid ◽  
Soybean Proteins ◽  
Yeast Two Hybrid System ◽  
Two Hybrid ◽  
Virus Proteins

scholarly journals The Rotavirus Enterotoxin NSP4 Directly Interacts with the Caveolar Structural Protein Caveolin-1

2006 ◽  
Vol 80 (6) ◽  
pp. 2842-2854 ◽  
Author(s):  
Rebecca D. Parr ◽  
Stephen M. Storey ◽  
DeAnne M. Mitchell ◽  
Avery L. McIntosh ◽  
Minglong Zhou ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Nonstructural Protein ◽  
Direct Interaction ◽  
Cell Periphery ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Ussing Chambers ◽  
Caveolin 1 ◽  
Two Hybrid

ABSTRACT Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4114-135) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.

N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan

2009 ◽  
Vol 420 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Eriko Aoyama ◽  
Takako Hattori ◽  
Mitsuhiro Hoshijima ◽  
Daisuke Araki ◽  
Takashi Nishida ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Binding Protein ◽  
Direct Interaction ◽  
Tissue Growth ◽  
Yeast Two Hybrid ◽  
Ccn Family ◽  
Two Hybrid

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, as a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type 1 repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan, and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding In vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP–rVWC peptide effectively enhanced the production and release of aggrecan compared with the rTSP–rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC modules, and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

scholarly journals Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit, Requires Complex Formation of β4 and HD1/Plectin, and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

1998 ◽  
Vol 142 (1) ◽  
pp. 271-284 ◽  
Author(s):  
Roel Q.J. Schaapveld ◽  
Luca Borradori ◽  
Dirk Geerts ◽  
Manuel R. van Leusden ◽  
Ingrid Kuikman ◽  
...  
Keyword(s):  
Bullous Pemphigoid ◽  
Direct Interaction ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Fibronectin Type Iii ◽  
Activation Motif ◽  
Β4 Integrin ◽  
Two Hybrid

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.

Soybean (Glycine max) PHYTOCHROME-INTERACTION FACTOR 1 induced skotomorphogenesis and de-etiolation in Arabidopsis

2020 ◽  
pp. 1-12
Author(s):  
Tianliang Wang ◽  
Shuo Wang ◽  
Jinhao Zhang ◽  
Fan Yan ◽  
Yajing Liu ◽  
...  
Keyword(s):  
Glycine Max ◽  
Light Response ◽  
Chlorophyll Synthesis ◽  
Energy Utilization ◽  
Yeast Two Hybrid ◽  
Etiolated Seedlings ◽  
Photooxidative Damage ◽  
Interaction Factor ◽  
Glycine Max L ◽  
Two Hybrid

Skotomorphogenesis occurs after germination and before excavation in plants. It inhibits excessive absorbed energy in cells and can prevent the lethal photooxidative damage caused by transitioning from skotomorphogenesis to photomorphogenesis for light energy utilization. To investigate the mechanisms underlying photoreactions in soybean [Glycine max (L.) Merr.], we identified and isolated soybean phytochrome-interaction factor 1 (GmPIF1). A yeast two-hybrid (Y2H) assay showed that GmPIF1 interacted with photoactive PHYTOCHROME A (PHYA) and B (PHYB) in both soybean and Arabidopsis (GmPHYA, GmPHYB, AtPHYA, and AtPHYB). To analyze its function, we ectopically over-expressed GmPIF1 in wild type and pif1 mutant Arabidopsis. In etiolated seedlings, GmPIF1 caused hypocotyl elongation, cotyledon closed, apical hooks folded, and less accumulation of protochlorophyllide. In Y2H, GmPIF1 interacted with AtHDA15 that inhibited chlorophyll synthesis under dark conditions. After transition from darkness to white light, GmPIF1 promotes the reduction of photobleaching and induced de-etiolation. Moreover, GmPIF1 inhibited PHYA- and PHYB-mediated seed germination. Our findings increase our understanding of the regulatory network of light response in soybean and provide useful gene resources for soybean breeding in programs and genetics engineering.

scholarly journals Transcription Activator Swi6 Interacts with Mbp1 in MluI Cell Cycle Box-Binding Complex and Regulates Hyphal Differentiation and Virulence in Beauveria bassiana

2021 ◽  
Vol 7 (6) ◽  
pp. 411
Author(s):  
Jin-Li Ding ◽  
Jia Hou ◽  
Xiu-Hui Li ◽  
Ming-Guang Feng ◽  
Sheng-Hua Ying
Keyword(s):  
Cell Cycle ◽  
Direct Interaction ◽  
Hyphal Growth ◽  
Transcription Activator ◽  
Liquid Media ◽  
Yeast Two Hybrid ◽  
Genetic Pathways ◽  
Morphological Transitions ◽  
Lethal Time ◽  
Two Hybrid

Mbp1 protein acts as a DNA-binding protein in MluI cell cycle box-binding complex (MBF) and plays an essential role in filamentous myco-pathogen Beauveria bassiana.In the current study, BbSwi6 (a homologue of yeast Swi6) was functionally characterized in B.bassiana. Both BbSwi6 and BbMbp1 localize in the nucleus and display a direct interaction relationship which is indicated by a yeast two-hybrid assay. BbSwi6 significantly contributes to hyphal growth, asexual sporulation and virulence. On the aerial surface, ΔBbSwi6 grew slower on various nutrients and displayed abnormal conidia-producing structures, which hardly produced conidia. In liquid media, BbSwi6 loss led to 90% reduction in blastospore yield. Finally, the virulence of the ΔBbSwi6 mutant was modestly weakened with a reduction of 20% in median lethal time. Comparative transcriptomics revealed that BbSwi6 mediated different transcriptomes during fungal development into conidia and blastospores. Notably, under the indicated condition, the BbSwi6-mediated transcriptome significantly differed to that mediated by BbMbp1. Our results demonstrate that, in addition to their roles as the interactive components in MBF, BbSwi6 and BbMbp1 mediate divergent genetic pathways during morphological transitions in B. bassiana.

scholarly journals Direct interaction of β-dystroglycan with F-actin

2003 ◽  
Vol 375 (2) ◽  
pp. 329-337 ◽  
Author(s):  
Yun-Ju CHEN ◽  
Heather J. SPENCE ◽  
Jacqueline M. CAMERON ◽  
Thomas JESS ◽  
Jane L. ILSLEY ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
Yeast Two Hybrid ◽  
Glycoprotein Complex ◽  
Biological Studies ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein ◽  
Two Hybrid ◽  
Sarcolemmal Integrity

Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

scholarly journals Purification of the RelB and RelE Proteins ofEscherichia coli: RelE Binds to RelB and to Ribosomes

2001 ◽  
Vol 183 (8) ◽  
pp. 2700-2703 ◽  
Author(s):  
Cheryl Galvani ◽  
Jefferson Terry ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Binding Activity ◽  
In Vitro Assay ◽  
Ofescherichia Coli ◽  
Yeast Two Hybrid ◽  
Vitro Assay ◽  
Ribosome Binding ◽  
Two Hybrid

ABSTRACT The direct interaction of the Escherichia colicytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the relB andrelE genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.

scholarly journals Direct Binding of CRKL to BCR-ABL Is Not Required for BCR-ABL Transformation

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 297-306 ◽  
Author(s):  
Conor Heaney ◽  
Kathryn Kolibaba ◽  
Arun Bhat ◽  
Tsukasa Oda ◽  
Sayuri Ohno ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Direct Interaction ◽  
Myelogenous Leukemia ◽  
Indirect Interaction ◽  
Yeast Two Hybrid ◽  
Rich Region ◽  
Amino Terminal ◽  
C Terminus ◽  
Two Hybrid ◽  
Proline Rich Region

CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL. CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.

scholarly journals Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

1998 ◽  
Vol 72 (8) ◽  
pp. 6732-6741 ◽  
Author(s):  
Wenkai Xiang ◽  
Andrea Cuconati ◽  
Debra Hope ◽  
Karla Kirkegaard ◽  
Eckard Wimmer
Keyword(s):  
Hybrid System ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Genome Replication ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

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