scholarly journals An E3 ubiquitin ligase fromNicotiana benthamianatargets the replicase ofBamboo mosaic virusand restricts its replication

2018 ◽  
Author(s):  
I-Hsuan Chen ◽  
Jui-En Chang ◽  
Chen-Yu Wu ◽  
Ying-Ping Huang ◽  
Yau-Huei Hsu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Tobacco Rattle Virus ◽  
E3 Ubiquitin Ligase ◽  
Infection Process ◽  
Ring Domain ◽  
Virus Induced Gene Silencing ◽  
Ubiquitin E3 Ligase ◽  
Rapid Amplification

AbstractOne upregulated host gene identified previously was found involved in the infection process ofBamboo mosaic virus(BaMV). The full-length cDNA of this gene was cloned by 5′- and 3′-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved RING-domain and a transmembrane domain. The gene might function as an E3 ubiquitin ligase. We designated this protein inNicotiana benthamianaas ubiquitin E3 ligase containing RING-domain 1 (NbUbE3R1). Further characterization by usingTobacco rattle virus-based virus-induced gene silencing revealed an increased BaMV accumulation in both knockdown plants and protoplasts. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed inN. benthamianaplants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting BaMV accumulation. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an upregulated gene, NbUbE3R1, that plays a role in BaMV replication.

scholarly journals The RBCC GeneRFP2(Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD

2007 ◽  
Vol 18 (5) ◽  
pp. 1670-1682 ◽  
Author(s):  
Mikael Lerner ◽  
Martin Corcoran ◽  
Diana Cepeda ◽  
Michael L. Nielsen ◽  
Roman Zubarev ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Yellow Fluorescent Protein ◽  
E3 Ubiquitin Ligase ◽  
Coiled Coil ◽  
Ring Finger ◽  
Ubiquitin Ligase Activity

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

2021 ◽  
Author(s):  
◽  
Carla Coppola
Keyword(s):  
Ubiquitin Ligase ◽  
Physcomitrella Patens ◽  
Fluorescent Protein ◽  
Storage Proteins ◽  
Higher Plants ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligases ◽  
Ubiquitin Ligase Activity ◽  
Vacuolar Transport

In this study, I focused on a new family of receptors, called RMRs (Receptor-like Membrane RING-H2) and I tried to investigate their role in the moss Physcomitrium patens Mitten (previously Physcomitrella patens). There is some evidence that in Angiosperms, RMRs are vacuolar receptors for the neutral/storage vacuole that is a compartment where storage proteins and metabolites are accumulated during seeds development or in somatic tissues. It is distinguished from lytic vacuole which has the same functions as animal lysosomes. The five PpRMR genes have been knocked-out, yielding viable material without visible phenotype (Ayachi, 2012). A trafficking phenotype was described by Fahr (2017) who generated the construct Citrine-Cardosin (Ci-Card) composed of the fluorescent protein Citrine fused to the C-terminal vacuolar sorting determinant (ctVSD) from cardosin A (cardosin is addressed to the vacuole in higher plants —Pereira et al., 2013). The fusion protein was delivered to the central vacuole of PpWT but mistargeted in PpRMR-KO lines, indicating that the targeting of this protein to the vacuole depends on PpRMRs. The introduction of this thesis presents the plant endomembrane system, with particular attention to vacuolar transport and ubiquitylation. In the second chapter, I show the techniques used to attempt to detect PpRMRs by Western Blot: our failure may be due to a rapid degradation of these proteins, which could prevent their detection. In the third chapter, I focused on PpRMR2 involvement in ubiquitylation. We hypothesize that PpRMRs are E3 ligases because they are members of the PA-TM-RING protein family. Most of these proteins have an E3 ubiquitin ligase activity in animals (Seroogy et al., 2004; Borchers et al., 2002), for this reason, we think that plant PpRMRs could have this function as well, which could contribute to vacuolar targeting. Indeed, I could confirm that PpRMR2 has an E3 ubiquitin ligase activity. PpRMRs substrates are still unknown in moss thus we have analysed putative candidates supposing that they could be ubiquitylated by PpRMRs. We have tested this hypothesis through in vitro ubiquitylation assays, obtaining ambiguous results. In the fourth chapter, I show preliminary results about the visible phenotype of PpRMR-KO mutants: PpWT and PpRMR-KO lines displayed phenotypic differences in leafy gametophores, which were accentuated upon salt stress exposure. Lastly, I transformed the transgenic lines PpWT/Ci-Card and Pp5KO/Ci-Card with mutated versions of PpRMR2 and analysed their effect on vacuolar transport by confocal microscopy. For most of the constructions tested, the trafficking was perturbed in both lines. Only PpWT/Ci-Card expressing PpRMR2ΔSer (lacking the Serine-Rich motif) displayed a typical vacuolar pattern.

scholarly journals Structure and function of E3 ubiquitin ligase mindbomb RING domain

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Robert D Wardlow ◽  
Sung Hee Choi ◽  
Brian McMillan ◽  
Stephen C Blacklow
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structure And Function ◽  
Ring Domain ◽  
And Function

scholarly journals Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

2021 ◽  
Vol 320 (1) ◽  
pp. C45-C56
Author(s):  
David C. Hughes ◽  
Daniel C. Turner ◽  
Leslie M. Baehr ◽  
Robert A. Seaborne ◽  
Mark Viggars ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Tibialis Anterior Muscle ◽  
E3 Ubiquitin Ligase ◽  
Skeletal Muscle Atrophy ◽  
The Impact

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5’s role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3β/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (−9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3β activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (−4.6%) and larger reductions in fiber CSA (−18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

scholarly journals AtPPRT1, an E3 Ubiquitin Ligase, Enhances the Thermotolerance in Arabidopsis

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1074
Author(s):  
Yu Liu ◽  
Shuya Xiao ◽  
Haoran Sun ◽  
Linsen Pei ◽  
Yingying Liu ◽  
...  
Keyword(s):  
Heat Stress ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Degradation Pathway ◽  
Vital Role ◽  
Loss Of Function ◽  
Positive Role ◽  
Heat Stress Response ◽  
Acquired Thermotolerance ◽  
Ubiquitin E3 Ligase

E3 ubiquitin ligase plays a vital role in the ubiquitin-mediated heat-related protein degradation pathway. Herein, we report that the expression of AtPPRT1, a C3HC4 zinc-finger ubiquitin E3 ligase gene, was induced by heat stress, and the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity after basal and acquired thermotolerance. To further explore the function of AtPPRT1 in heat stress response (HSR), we used the atpprt1 mutant and AtPPRT1-overexpressing lines (OE2 and OE10) to expose in heat shock. In this study, the atpprt1 mutant had a lower germination and survival rate than those of Col-0 when suffered from the heat stress, whereas OEs enhanced basal and acquired thermotolerance in Arabidopsis seedlings. When compared to Col-0 and OEs, loss-of-function in AtPPRT1 resulted in lower chlorophyll retention and higher content of reactive oxygen species (ROS) after heat treatment. Moreover, the transcript levels of AtPPRT1 and several heat-related genes (AtZAT12, AtHSP21 and AtHSFA7a) were upregulated to greater extents in OEs and lower extents in atpprt1 compared to Col-0 after heat treated. Hence, we suggest that AtPPRT1 may act as a positive role in regulating the high temperature by mediating the degradation of unknown target proteins.

scholarly journals Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

2019 ◽  
Vol 20 (16) ◽  
pp. 3976 ◽  
Author(s):  
Hongqiu Zeng ◽  
Yanwei Xie ◽  
Guoyin Liu ◽  
Yunxie Wei ◽  
Wei Hu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Functional Genomics ◽  
Transient Expression ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Mosaic Disease ◽  
African Cassava Mosaic Virus ◽  
Cassava Mosaic Disease ◽  
Virus Induced Gene Silencing ◽  
Transient Expression Assay

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

scholarly journals TaClpS1, Negatively Regulates Wheat Resistance Against Puccinia striiformis f. sp. tritici

2020 ◽  
Author(s):  
Qian Yang ◽  
Md Ashraful Islam ◽  
Kunyan Cai ◽  
Shuxin Tian ◽  
Yan Liu ◽  
...  
Keyword(s):  
Immune Response ◽  
Ubiquitin Ligase ◽  
Puccinia Striiformis ◽  
E3 Ubiquitin Ligase ◽  
Transit Peptide ◽  
26S Proteasome ◽  
Plant Responses ◽  
Virus Induced Gene Silencing ◽  
Chloroplast Transit Peptide

Abstract Background: The degradation of intracellular proteins plays an essential role in plant responses to stressful environments. ClpS1 and E3 ubiquitin ligase function as adaptors for selecting target substrates in caseinolytic peptidase (Clp) proteases pathways and the 26S proteasome system, respectively. Currently, the role of E3 ubiquitin ligase in the plant immune response to pathogens is well defined. However, the role of ClpS1 in the plant immune response to pathogens remains unknown. Results: Here, we identified and characterized wheat (Triticum aestivum) ClpS1 (TaClpS1). TaClpS1 encoded 161 amino acids, contained a conserved ClpS domain and a chloroplast transit peptide (1-32 aa). TaClpS1 was found to be specifically localized in the chloroplast when expressed transiently in wheat protoplasts. The transcript level of TaClpS1 in wheat was significantly induced during infection by Puccinia striiformis f. sp. tritici (Pst). Knock-down of TaClpS1 via virus-induced gene silencing (VIGS) resulted in an increase in resistance against Pst, accompanied by an increase in the hypersensitive response (HR), accumulation of reactive oxygen species (ROS) and expression of TaPR1 and TaPR2, and a reduction in the growth of Pst. Furthermore, heterologous expression of TaClpS1 in Nicotiana benthamiana enhanced the infection by Phytophthora parasitica. Conclusions: These results suggest that TaClpS1 negatively regulates the resistance of wheat to Pst.

scholarly journals TaClpS1, Negatively Regulates Wheat Resistance Against Puccinia striiformis f. sp. tritici

2020 ◽  
Author(s):  
Qian Yang ◽  
Md Ashraful Islam ◽  
Kunyan Cai ◽  
Shuxin Tian ◽  
Yan Liu ◽  
...  
Keyword(s):  
Immune Response ◽  
Ubiquitin Ligase ◽  
Puccinia Striiformis ◽  
E3 Ubiquitin Ligase ◽  
Transit Peptide ◽  
26S Proteasome ◽  
Plant Responses ◽  
Virus Induced Gene Silencing ◽  
Chloroplast Transit Peptide

Abstract Background: The degradation of intracellular proteins plays an essential role in plant responses to stressful environments. ClpS1 and E3 ubiquitin ligase function as adaptors for selecting target substrates in caseinolytic peptidase (Clp) proteases pathways and the 26S proteasome system, respectively. Currently, the role of E3 ubiquitin ligase in the plant immune response to pathogens is well defined. However, the role of ClpS1 in the plant immune response to pathogens remains unknown. Results: Here, wheat (Triticum aestivum) ClpS1 (TaClpS1) was studied and resulted to encode 161 amino acids, containing a conserved ClpS domain and a chloroplast transit peptide (1-32 aa). TaClpS1 was found to be specifically localized in the chloroplast when expressed transiently in wheat protoplasts. The transcript level of TaClpS1 in wheat was significantly induced during infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaClpS1 via virus-induced gene silencing (VIGS) resulted in an increase in wheat resistance against Pst, accompanied by an increase in the hypersensitive response (HR), accumulation of reactive oxygen species (ROS) and expression of TaPR1 and TaPR2, and a reduction in the number of haustoria, length of infection hypha and infection area of Pst. Furthermore, heterologous expression of TaClpS1 in Nicotiana benthamiana enhanced the infection by Phytophthora parasitica. Conclusions: These results suggest that TaClpS1 negatively regulates the resistance of wheat to Pst.

scholarly journals TaClpS1, Negatively Regulates Wheat Resistance Against Puccinia striiformis f. sp. tritici

2020 ◽  
Author(s):  
Qian Yang ◽  
Md Ashraful Islam ◽  
Kunyan Cai ◽  
Shuxin Tian ◽  
Yan Liu ◽  
...  
Keyword(s):  
Immune Response ◽  
Ubiquitin Ligase ◽  
Puccinia Striiformis ◽  
E3 Ubiquitin Ligase ◽  
Transit Peptide ◽  
26S Proteasome ◽  
Plant Responses ◽  
Virus Induced Gene Silencing ◽  
Chloroplast Transit Peptide

Abstract Background: The degradation of intracellular proteins plays an essential role in plant responses to stressful environments. ClpS1 and E3 ubiquitin ligase function as adaptors for selecting target substrates in caseinolytic peptidase (Clp) proteases pathways and the 26S proteasome system, respectively. Currently, the role of E3 ubiquitin ligase in the plant immune response to pathogens is well defined. However, the role of ClpS1 in the plant immune response to pathogens remains unknown. Results: Here, wheat (Triticum aestivum) ClpS1 (TaClpS1) was studied and resulted to encode 161 amino acids, containing a conserved ClpS domain and a chloroplast transit peptide (1-32 aa). TaClpS1 was found to be specifically localized in the chloroplast when expressed transiently in wheat protoplasts. The transcript level of TaClpS1 in wheat was significantly induced during infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaClpS1 via virus-induced gene silencing (VIGS) resulted in an increase in wheat resistance against Pst, accompanied by an increase in the hypersensitive response (HR), accumulation of reactive oxygen species (ROS) and expression of TaPR1 and TaPR2, and a reduction in the number of haustoria, length of infection hypha and infection area of Pst. Furthermore, heterologous expression of TaClpS1 in Nicotiana benthamiana enhanced the infection by Phytophthora parasitica. Conclusions: These results suggest that TaClpS1 negatively regulates the resistance of wheat to Pst.

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