scholarly journals A Host-Factor Interaction and Localization Map for a Plant-Adapted Rhabdovirus Implicates Cytoplasm-Tethered Transcription Activators in Cell-to-Cell Movement

2010 ◽  
Vol 23 (11) ◽  
pp. 1420-1432 ◽  
Author(s):  
Byoung-Eun Min ◽  
Kathleen Martin ◽  
Renyuan Wang ◽  
Petra Tafelmeyer ◽  
Max Bridges ◽  
...  
Keyword(s):  
Cell Movement ◽  
Host Factors ◽  
Bimolecular Fluorescence Complementation ◽  
Binary Interaction ◽  
Transcription Activator ◽  
Nuclear Localization Signals ◽  
Putative Movement Protein ◽  
Interaction Map ◽  
Transcription Activators ◽  
Two Hybrid

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

scholarly journals Identification of a Plum pox virus CI-Interacting Protein from Chloroplast That Has a Negative Effect in Virus Infection

2006 ◽  
Vol 19 (3) ◽  
pp. 350-358 ◽  
Author(s):  
I. Jiménez ◽  
L. López ◽  
J. M. Alamillo ◽  
A. Valli ◽  
J. A. García
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Movement ◽  
Host Factors ◽  
Plum Pox Virus ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Yeast Two Hybrid System ◽  
Negative Effect ◽  
Virus Interactions ◽  
Two Hybrid

The cylindrical inclusion (CI) protein of potyviruses is involved in virus replication and cell-to-cell movement. These two processes should rely on multiple plant-virus interactions; however, little is known about the host factors that are involved in, or that may interfere with, CI functions. By using a yeast two-hybrid system, the CI protein from Plum pox virus (PPV) was found to interact with the photosystem I PSI-K protein, the product of the gene psaK, of Nicotiana benthamiana. Coexpression of PPV CI was shown to cause a decrease in the accumulation level of PSI-K transiently expressed in N. benthamiana leaves. To test the biological relevance of this interaction, we have analyzed the infection of PPV in N. benthamiana plants in which psaK gene expression has been silenced by RNA interference, as well as in Arabidopsis thaliana psaK knockout plants. Our results show that downregulation of the psaK gene leads to higher PPV accumulation, suggesting a role for the CI-PSI-K interaction in PPV infection.

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.

Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2427-2435 ◽  
Author(s):  
F.L. Conlon ◽  
S.G. Sedgwick ◽  
K.M. Weston ◽  
J.C. Smith
Keyword(s):  
Transcription Activation ◽  
Zebrafish Embryos ◽  
Transcription Activator ◽  
Activation Domain ◽  
Activation Domains ◽  
Dorsal Mesoderm ◽  
Transcription Activators ◽  
Carboxy Terminal ◽  
Genetic Mutants ◽  
Axial Development

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

Characterization of a Novel Barley Protein, HCP1, That Interacts with the Brome mosaic virus Coat Protein

2003 ◽  
Vol 16 (4) ◽  
pp. 352-359 ◽  
Author(s):  
Yasushi Okinaka ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno ◽  
Iwao Furusawa
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Brome Mosaic Virus ◽  
Host Factors ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Long Distance ◽  
C Terminus ◽  
Barley Protein ◽  
Two Hybrid

Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants. This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components. To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 × 106 independent clones, using a yeast two-hybrid system. Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated. One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1. HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues. A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1. Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley. These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.

scholarly journals Wheat Yellow Mosaic Virus NIb Interacting with Host Light Induced Protein (LIP) Facilitates Its Infection through Perturbing the Abscisic Acid Pathway in Wheat

Biology ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 80 ◽  
Author(s):  
Zhang ◽  
Liu ◽  
Zhong ◽  
Zhang ◽  
Xu ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Mosaic Virus ◽  
Distribution Patterns ◽  
Host Factors ◽  
Bimolecular Fluorescence Complementation ◽  
Yellow Mosaic Virus ◽  
Wheat Yellow Mosaic Virus ◽  
Positive Sense ◽  
Acid Pathway ◽  
Coding Capacity

Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly reliant on host factors to fulfill their infection. However, few host factors have been identified to participate in wheat yellow mosaic virus (WYMV) infection. Here, we demonstrate that wheat (Triticum aestivum) light-induced protein (TaLIP) interacts with the WYMV nuclear inclusion b protein (NIb). A bimolecular fluorescence complementation (BIFC) assay displayed that the subcellular distribution patterns of TaLIP were altered by NIb in Nicotiana benthamiana. Transcription of TaLIP was significantly decreased by WYMV infection and TaLIP-silencing wheat plants displayed more susceptibility to WYMV in comparison with the control plants, suggesting that knockdown of TaLIP impaired host resistance. Moreover, the transcription level of TaLIP was induced by exogenous abscisic acid (ABA) stimuli in wheat, while knockdown of TaLIP significantly repressed the expression of ABA-related genes such as wheat abscisic acid insensitive 5 (TaABI5), abscisic acid insensitive 8 (TaABI8), pyrabatin resistance 1-Llike (TaPYL1), and pyrabatin resistance 3-Llike (TaPYL3). Collectively, our results suggest that the interaction of NIb with TaLIP facilitated the virus infection possibly by disturbing the ABA signaling pathway in wheat.

scholarly journals C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking

2011 ◽  
Vol 22 (12) ◽  
pp. 2083-2093 ◽  
Author(s):  
P. James Scrivens ◽  
Baraa Noueihed ◽  
Nassim Shahrzad ◽  
Sokunthear Hul ◽  
Stephanie Brunet ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Multidisciplinary Approach ◽  
Early Stage ◽  
Binary Interaction ◽  
The Novel ◽  
Novel Proteins ◽  
Bona Fide ◽  
Interaction Map ◽  
Equivalent Complex

TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I–equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.

CsSWEET1a and CsSWEET17 Mediate Growth and Freezing Tolerance by Promoting Sugar Transport across the Plasma Membrane

2020 ◽  
Vol 61 (9) ◽  
pp. 1669-1682
Author(s):  
Lina Yao ◽  
Changqing Ding ◽  
Xinyuan Hao ◽  
Jianming Zeng ◽  
Yajun Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Freezing Tolerance ◽  
Sugar Transport ◽  
Bimolecular Fluorescence Complementation ◽  
Freezing Stress ◽  
Sugar Uptake ◽  
Wild Type ◽  
Alternatively Spliced ◽  
Germination And Growth ◽  
Two Hybrid

Abstract Sugars Will Eventually be Exported Transporters (SWEETs) are important in plant biological processes. Expression levels of CsSWEET1a and CsSWEET17 are induced by cold acclimation (CA) and cold stress in Camellia sinensis. Here, we found that CsSWEET17 was alternatively spliced, and its exclusion (Ex) transcript was associated with the CA process. Both plasma membrane-localized CsSWEET1a and CsSWEET17 transport hexoses, but cytoplasm-localized CsSWEET17-Ex does not. These results indicate that alternative splicing may be involved in regulating the function of SWEET transporters in response to low temperature in plants. The extra C-terminal of CsSWEET17, which is not found in the tonoplast fructose transporter AtSWEET17, did not affect its plasma membrane localization but promoted its sugar transport activities. The overexpression (OE) of CsSWEET1a and CsSWEET17 genes resulted in an increased sugar uptake in Arabidopsis, affecting plant germination and growth. The leaf and seed sizes of the CsSWEET17-OE lines were significantly larger than those of the wild type. Moreover, the OE of CsSWEET1a and CsSWEET17 significantly reduced the relative electrolyte leakage levels under freezing stress. Compared with the wild type, the expression of AtCWINV genes was suppressed in both CsSWEET1a-OE and CsSWEET17-OE lines, indicating the alteration in sugar contents in the cell walls of the OE lines. Furthermore, the interaction between CsSWEET1a and CsSWEET17 was confirmed using yeast two-hybrid and bimolecular fluorescence complementation assays. We showed that CsSWEET1a and CsSWEET17 form homo-/heterodimers in the plasma membrane and mediate the partitioning of sugars between the cytoplasm and the apoplast, thereby regulating plant growth and freezing tolerance.

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 The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg–HCPro and VPg–VPg interactions

2003 ◽  
Vol 84 (10) ◽  
pp. 2861-2869 ◽  
Author(s):  
Ma. Leonora M. Yambao ◽  
Chikara Masuta ◽  
Kenji Nakahara ◽  
Ichiro Uyeda
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Yellow Vein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Clover Yellow Vein Virus ◽  
Two Hybrid ◽  
Far Western Blot

Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro–VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro–VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro–VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg–VPg interaction and the central 19 amino acids were needed for the HCPro–VPg interaction.

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