scholarly journals The necrotrophic effector ToxA from Parastagonospora nodorum interacts with wheat NHL proteins to facilitate Tsn1-mediated necrosis

2021 ◽  
Author(s):  
Bayantes Dagvadorj ◽  
Megan A. Outram ◽  
Simon J. Williams ◽  
Peter S. Solomon
Keyword(s):  
Cell Death ◽  
Confocal Microscopy ◽  
Molecular Mechanisms ◽  
Wheat Cultivars ◽  
In Planta ◽  
Yeast Two Hybrid ◽  
Induce Cell Death ◽  
Parastagonospora Nodorum ◽  
Necrotrophic Effector ◽  
Two Hybrid

SummaryThe plant pathogen Parastagonospora nodorum secretes necrotrophic effectors to promote disease. These effectors induce cell death on wheat cultivars carrying dominant susceptibility genes in an inverse gene-for-gene manner. However, the molecular mechanisms underpinning these interactions and resulting cell death remain unclear. Here, we used a yeast-two-hybrid library approach to identify wheat proteins that interact with the necrotrophic effector ToxA. Using this strategy, we identified an interaction between ToxA and a wheat transmembrane NDR/HIN1-like protein (TaNHL10) and confirmed the interaction using in-planta co-immunoprecipitation and confocal microscopy co-localization analysis. We showed that the C-terminus of TaNHL10 is extracellular whilst the N-terminus was localized in the cytoplasm. Further analyses using yeast-two-hybrid and confocal microscopy co-localization showed that ToxA interacts with the C-terminal LEA2 extracellular domain of TaNHL10. Random mutagenesis was then used to identify a ToxA mutant, ToxAN109D, which was unable to interact with TaNHL10 in yeast-two-hybrid assays. Subsequent heterologous expression and purification of ToxAN109D in Nicotiania benthamiana revealed that the mutated protein was unable to induce necrosis on Tsn1-dominant wheat cultivars confirming that the interaction of ToxA with TaNHL10 is required to induce cell death. Collectively, these data advance our understanding on how ToxA induces cell death during infection and further highlights the importance of host cell surface interactions in necrotrophic pathosystems.

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

A Cell-Death-Defying Factor, Anamorsin, Contributes Cell Growth through Binding with PICOT and Inactivation of PKCs and p38MAPK.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1552-1552
Author(s):  
Hirohiko Shibayama ◽  
Yuri Saito ◽  
Akira Tanimura ◽  
Hirokazu Tanaka ◽  
Itaru Matsumura ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Cyclin D1 ◽  
P38 Mapk ◽  
Conflicts Of Interest ◽  
Biological Effects ◽  
Terminal Region ◽  
Yeast Two Hybrid ◽  
A Cell ◽  
Two Hybrid

Abstract Abstract 1552 [Background] Anamorsin (also called CIAPIN-1) is a cell-death-defying factor, which was originally isolated as a molecule that conferred resistance to apoptosis induced by growth factor starvation. Anamorsin is ubiquitously expressed in various organs, including hematopoietic tissues like bone marrow, spleen, and thymus. Anamorsin-deficient (AM KO) mice die in late gestation. AM KO embryos are anemic and the size of the embryos is very small. It is thought that anamorsin plays a crucial role in hematopoiesis during late and/or terminal stages of differentiation and embryogenesis. Anamorsin does not show any homology to known apoptosis and cell growth regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. (J Exp Med 199: 581–592, 2004) Anamorsin is composed of a methyltransferase domain in the N-terminal region and a hypothetical Zn-ribbon-like motif in the C-terminal region, however, the precise biological effects of anamorsin remained to be elucidated. [Methods & Results] In an attempt to clarify the mechanisms of the anamorsin functions, we have performed the yeast-two-hybrid assay to identify anamorsin-interacting molecules and found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to anamorsin. Next, we tried to determine the binding sites of anamorsin and PICOT with the yeast-two-hybrid assays by using their several deletion mutants and found that the N-terminal region (11-180aa) of anamorsin and the N-terminal region (18-117aa) of PICOT were essential for binding each other. Furthermore, we tried to examine the signaling pathways by using murine embryonic fibroblast (MEF) cells produced from E-14.5 AM KO or wild type (WT) embryos. The proliferation of AM KO MEF cells was quite retarded compared with that of WT MEF cells. It is found that the phosphorylation states of ERK1/2, NFkB, and AKT were similar both in AM KO MEF cells and WT MEF cells, while PKCθ, PKCδ and p38 MAPK were more phosphorylated in AM KO MEF cells than in WT MEF cells. The expression of cyclin D1, the target molecule of p38 MAPK, was down-regulated in AM KO MEF cells. The PKC inhibitor, Rottlerin, blocked p38 MAPK phosphorylation and the p38 MAPK inhibitor, SB203580, restored the expression of cyclin D1 and the cell growth of AM KO MEF cells. [Conclusion] P38 MAPK, the stress activated MAPK, and PKCs have been known to link to cell growth, differentiation, and apoptosis, and also to be essential for cell survival in response to various stimuli. From our results, it was thought that PKCθ, PKCδ, and p38 MAPK activation lead to cell cycle retardation of AM KO MEF cells and anamorsin might negatively regulate PKCθ, PKCδ, and p38 MAPK cooperatively with PICOT in MEF cells. This study clarified a novel mechanism of the anamorsin functions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Proteasomal degradation of JAZ9 by SALT- AND DROUGHT-INDUCED RING FINGER1 during pathogen infection

2021 ◽  
Author(s):  
Vemanna Ramu ◽  
Garima Pal ◽  
Sunhee Oh ◽  
Kirankumar S Mysore
Keyword(s):  
Ubiquitin Ligase ◽  
Disease Susceptibility ◽  
Plant Immunity ◽  
E3 Ubiquitin Ligase ◽  
In Planta ◽  
Yeast Two Hybrid ◽  
Pathogen Infection ◽  
Jaz Proteins ◽  
Molecular Fluorescence ◽  
Two Hybrid

E3 ubiquitin ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) plays a novel role in modulating plant immunity against pathogens. The molecular interactors of SDIR1 during pathogen infection are not known. SDIR1 interacting Jasmonate ZIM-domain (JAZ) proteins were identified through a yeast two-hybrid (Y2H) screen. Full length JAZ9 interacts with SDIR1 only in the presence of coronatine, a bacteria secreted toxin, or jasmonic acid (JA) in Y2H assay. The bi-molecular fluorescence complementation and pull-down assays confirm the in planta interaction of these proteins. JAZ9 proteins, negative regulators of JA-mediated plant defense, were degraded during the pathogen infection by SDIR1 through a proteasomal pathway causing disease susceptibility against hemibiotrophic pathogens.

scholarly journals Interferon Inducible Transmembrane Protein 3 Inhibits Influenza Virus Replication and Inflammation by Interacting With ABHD16A

2020 ◽  
Author(s):  
Chen Liang ◽  
Limei Zhu ◽  
Jun Chen
Keyword(s):  
Influenza Virus ◽  
Confocal Microscopy ◽  
Virus Replication ◽  
Influenza Viruses ◽  
Expression Level ◽  
Hek293 Cells ◽  
Yeast Two Hybrid ◽  
Related Factors ◽  
Influenza Virus Replication ◽  
Two Hybrid

Abstract Background: Studies have shown that human interferon inducible transmembrane proteins (IFITM) family proteins have broad-spectrum antiviral capabilities. Preliminary studies in our laboratory have preliminarily proved that IFITMs have the effect of inhibiting influenza viruses. In order to further study its mechanism and role in the occurrence and development of influenza, relevant studies have been carried out.Methods: Fluorescence quantitative polymerase chain reaction (PCR) detection, yeast two-hybrid test and optical confocal microscopy were used to investigate the effect of hIFITM3 on influenza virus replication, the interaction with human abhydrolase domain containing 16A (hABHD16A) and the expression of inflammation-related factors.Results: In HEK293 cells, overexpression of hIFITM3 protein significantly inhibited the replication of influenza virus at 24h, 48h, and 72h; yeast two-hybrid experiment proved that IFITM3 interacts with ABHD16A; laser confocal microscopy observations showed that IFITM3 and ABHD16A co-localized in cell membrane area; the expression level of inflammation-related factors in cells overexpressing hIFITM3 or hABHD16A was detected by fluorescence quantitative PCR, and the results showed that the mRNA levels of interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-a and cyclooxygenase 2 (COX2) were significantly increased . But when IFITM3/ABHD16A was co-expressed, the mRNA expression levels of these cytokines were significantly reduced except for COX2. When influenza virus infected cells co-expressing IFITM3/ABHD16A, the expression level of inflammatory factors decreased compared with the control group, indicating that IFITM3 can play an important role in regulating inflammation balance.Conclusions: This study confirmed that hIFITM3 has an effect of inhibiting influenza virus replication. Furthermore, it was found that hIFITM3 interacts with hABHD16A, following which it can better inhibit the replication of influenza virus and the inflammatory response caused by the disease process.

scholarly journals Human Interferon Inducible Transmembrane Protein 3 (IFITM3) Inhibits Influenza Virus A Replication and Inflammation by Interacting with ABHD16A

2020 ◽  
Author(s):  
Liang Chen ◽  
Zhu Limei ◽  
Chen Jun
Keyword(s):  
Influenza Virus ◽  
Confocal Microscopy ◽  
Influenza Viruses ◽  
Expression Level ◽  
Hek293 Cells ◽  
Human Interferon ◽  
Yeast Two Hybrid ◽  
Related Factors ◽  
Influenza Virus A ◽  
Two Hybrid

Abstract Background: Studies have shown that human interferon inducible transmembrane proteins (hIFITMs) family proteins have broad-spectrum antiviral capabilities. Preliminary studies in our laboratory have preliminarily proved that hIFITMs have the effect of inhibiting influenza viruses. In order to further study its mechanism and role in the occurrence and development of influenza A, relevant studies have relevant studies have been carried out.Methods: Fluorescence quantitative polymerase chain reaction (PCR) detection, yeast two-hybrid test and optical confocal microscopy were used to investigate the effect of hIFITM3 on influenza virus A (IVA) replication, the interaction with human abhydrolase domain containing 16A (hABHD16A) and the expression of inflammation-related factors.Results: In HEK293 cells, overexpression of hIFITM3 protein significantly inhibited the replication of IVA at 24h, 48h, and 72h; yeast two-hybrid experiment proved that hIFITM3 interacts with hABHD16A; laser confocal microscopy observations showed that hIFITM3 and hABHD16A co-localized in cell membrane area; the expression level of inflammation-related factors in cells overexpressing hIFITM3 or hABHD16A was detected by fluorescence quantitative PCR, and the results showed that the mRNA levels of interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-a and for cyclooxygenase 2 (COX2) were significantly increased. But when hIFITM3/hABHD16A was co-expressed, the mRNA expression levels of these cytokines were significantly reduced except COX2. When influenza virus infected cells co-expressing hIFITM3/hABHD16A, the expression level of inflammatory factors decreased compared with the control group, indicating that hIFITM3 can play an important role in regulating inflammation balance.Conclusions: This study confirmed that hIFITM3 has an effect of inhibiting IVA replication. Furthermore, it was found that hIFITM3 interacts with hABHD16A, following which it can better inhibit the replication of influenza virus and the inflammatory response caused by the disease process.

scholarly journals Host targets of effectors of the lettuce downy mildew Bremia lactucae from cDNA-based yeast two-hybrid screening

2019 ◽  
Author(s):  
Alexandra J.E. Pelgrom ◽  
Claudia-Nicole Meisrimler ◽  
Joyce Elberse ◽  
Thijs Koorman ◽  
Mike Boxem ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Pathogenic Bacteria ◽  
Plant Pathogens ◽  
Molecular Mechanisms ◽  
Effector Proteins ◽  
Bremia Lactucae ◽  
Yeast Two Hybrid ◽  
Target Proteins ◽  
Successful Infection ◽  
Two Hybrid

AbstractPlant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped a network of physical interactions between B. lactucae effectors and lettuce target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten targets based on the number of independent colonies identified in the Y2H and two targets that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged target proteins and their interacting effectors. Importantly, relocalization of effectors or targets to the nucleus was observed for four effector-target pairs upon their co-expression, supporting their interaction in planta.

scholarly journals Interaction of porcine circovirus-like virus P1 capsid protein with host proteins

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Libin Wen ◽  
Jiaping Zhu ◽  
Fengxi Zhang ◽  
Qi Xiao ◽  
Jianping Xie ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Underlying Mechanism ◽  
Yeast Cells ◽  
Cellular Proteins ◽  
Porcine Circovirus ◽  
Yeast Two Hybrid ◽  
Host Proteins ◽  
Host Interactions ◽  
Wasting Syndrome ◽  
Two Hybrid

Abstract Background Porcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue. Results Five cellular proteins (EEP, Ral GDS, Bcl-2-L-12, CPS1, and one not identified) were found to interact with P1 Cap. The interaction between Cap and Ral GDS was confirmed by co-immunoprecipitation. Conclusions Our data are likely to support the future investigation of the underlying mechanism of P1 infection and pathogenesis.

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 Wheat thioredoxin (TaTrxh1) associates with the RD19-like cysteine protease TaCP1 to defend against stripe rust fungus through the modulation of programmed cell death.

2020 ◽  
Author(s):  
Beibei Shi ◽  
Xinbei Zhao ◽  
Min Li ◽  
Zihui Dong ◽  
Qichao Yang ◽  
...  
Keyword(s):  
Cell Death ◽  
Stripe Rust ◽  
Cysteine Protease ◽  
Puccinia Striiformis ◽  
Rust Fungus ◽  
Stress Signaling ◽  
Yeast Two Hybrid ◽  
Cellular Redox ◽  
Ros Accumulation ◽  
Two Hybrid

Thioredoxins (Trxs) function within the antioxidant network through modulation of the redox reaction(s) involved in oxidative-stress signaling. Given their function in regulating cellular redox, Trx proteins also fulfill key roles in plant immune signaling. Here, TaTrxh1, encoding a subgroup h member of the thioredoxin family, was identified and cloned in wheat (Triticum aestivum), which was rapidly induced by Puccinia striiformis f. sp. tritici (Pst) invasion and salicylic acid (SA) treatment. Overexpression of TaTrxh1 in tobacco (Nicotiana benthamiana) induced cell death. Silencing of TaTrxh1 in wheat enhanced susceptibility to Pst in different aspects, including ROS accumulation and pathogen-responsive, or related, gene expression. Herein, we observed that the cellular concentration of SA was significantly reduced in TaTrxh1-silenced plants, indicating that TaTrxh1 possibly regulates wheat resistance to stripe rust through a SA-associated defense signaling pathway. Using a yeast two-hybrid screen to identify TaTrxh1-interacting partners, we further show that interaction with TaCP1 (a RD19-like cysteine protease), and subsequent silencing of TaCP1 reduced wheat resistance to Pst. In total, the data presented herein demonstrate that TaTrxh1 enhances wheat resistance against Pst via SA-dependent resistance signaling and that TaTrxh1 interaction with TaCP1 is required for wheat resistance to stripe rust.

scholarly journals Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx

2002 ◽  
Vol 83 (4) ◽  
pp. 759-766 ◽  
Author(s):  
Xiao-Dong Li ◽  
Tomi P. Mäkelä ◽  
Deyin Guo ◽  
Rabah Soliymani ◽  
Vesa Koistinen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Nuclear Localization Signal ◽  
Nucleocapsid Protein ◽  
Molecular Mechanisms ◽  
Cellular Proteins ◽  
Yeast Two Hybrid ◽  
Localization Signal ◽  
Carboxyl Terminal ◽  
Two Hybrid ◽  
Hybrid Screening

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

Sign in / Sign up

Export Citation Format

Share Document