scholarly journals A comparative approach expands the protein–protein interaction node of the immune receptor XA21 in wheat and rice

Genome ◽  
2013 ◽  
Vol 56 (6) ◽  
pp. 315-326 ◽  
Author(s):  
Baoju Yang ◽  
Randy Ruan ◽  
Dario Cantu ◽  
Xiaodong Wang ◽  
Wanquan Ji ◽  
...  
Keyword(s):  
Signal Transduction Pathway ◽  
Full Length ◽  
Bimolecular Fluorescence Complementation ◽  
Comparative Approach ◽  
The Novel ◽  
Yeast Two Hybrid ◽  
Immune Receptor ◽  
Protein Protein Interaction ◽  
Novel Interactions ◽  
Two Hybrid

The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a nonsyntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that interspecific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals OFP1 Interaction with ATH1 Regulates Stem Growth, Flowering Time and Flower Basal Boundary Formation in Arabidopsis

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 399 ◽  
Author(s):  
Liguo Zhang ◽  
Lili Sun ◽  
Xiaofei Zhang ◽  
Shuquan Zhang ◽  
Dongwei Xie ◽  
...  
Keyword(s):  
Flowering Time ◽  
Large Scale ◽  
Stem Elongation ◽  
Stem Growth ◽  
Bimolecular Fluorescence Complementation ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
And Control ◽  
Two Hybrid

Ovate Family Protein1 (OFP1) is a regulator, and it is suspected to be involved in plant growth and development. Meanwhile, Arabidopsis Thaliana Homeobox (ATH1), a BEL1-like homeodomain (HD) transcription factor, is known to be involved in regulating stem growth, flowering time and flower basal boundary development in Arabidopsis. Previous large-scale yeast two-hybrid studies suggest that ATH1 possibly interact with OFP1, but this interaction is yet unverified. In our study, the interaction of OFP1 with ATH1 was verified using a directional yeast two-hybrid system and bimolecular fluorescence complementation (BiFC). Our results also demonstrated that the OFP1-ATH1 interaction is mainly controlled by the HD domain of ATH1. Meanwhile, we found that ATH1 plays the role of transcriptional repressor to regulate plant development and that OFP1 can enhance ATH1 repression function. Regardless of the mechanism, a putative functional role of ATH1-OFP1 may be to regulate the expression of the both the GA20ox1 gene, which is involved in gibberellin (GA) biosynthesis and control of stem elongation, and the Flowering Locus C (FLC) gene, which inhibits transition to flowering. Ultimately, the regulatory functional mechanism of OFP1-ATH1 may be complicated and diverse according to our results, and this work lays groundwork for further understanding of a unique and important protein–protein interaction that influences flowering time, stem development, and flower basal boundary development in plants.

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

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.

A Method to Generate Full-Length cDNA Molecules from Yeast Two-Hybrid Clones and RACE Products

1999 ◽  
Vol 268 (1) ◽  
pp. 161-162 ◽  
Author(s):  
Charles S. Hemenway ◽  
Benjamin W. Halligan ◽  
Grahame C.D. Gould
Keyword(s):  
Full Length ◽  
Yeast Two Hybrid ◽  
Full Length Cdna ◽  
Hybrid Clones ◽  
Two Hybrid

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.

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