The Experts below are selected from a list of 330 Experts worldwide ranked by ideXlab platform
Yauheiu Hsu - One of the best experts on this subject based on the ideXlab platform.
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stable display of artificially long foreign antigens on chimeric Bamboo Mosaic Virus particles
Viruses, 2021Co-Authors: Tsunghsien Chen, Chin-wei Lee, Na-sheng Lin, Yumin Feng, Yauheiu HsuAbstract:Plant Viruses can be genetically modified to generate chimeric Virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo Mosaic Virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease Virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete Virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant Virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.
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the lipid transfer protein 1 from nicotiana benthamiana assists Bamboo Mosaic Virus accumulation
Viruses, 2020Co-Authors: Lingying Chiu, Yauheiu Hsu, I-hsuan Chen, Chinghsiu TsaiAbstract:Host factors play a pivotal role in regulating Virus infection. Uncovering the mechanism of how host factors are involved in Virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo Mosaic Virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.
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fusion of a novel native signal peptide enhanced the secretion and solubility of bioactive human interferon gamma glycoproteins in nicotiana benthamiana using the Bamboo Mosaic Virus based expression system
Frontiers in Plant Science, 2020Co-Authors: Minchao Jiang, Na-sheng Lin, Weili Hsu, Tsuiling Hsu, Yauheiu HsuAbstract:Plant Viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo Mosaic Virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of 'Ser-Pro' motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis Virus and Influenza Virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.
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molecular biology of Bamboo Mosaic Virus a type member of the potexVirus genus
2020Co-Authors: Yauheiu Hsu, Chinghsiu Tsai, Na-sheng LinAbstract:The flexible filamentous plant Viruses are responsible for more than half of all agricultural loss worldwide. PotexVirus is one of the two most important flexible filamentous plant Viruses. Bamboo Mosaic Virus (BaMV), a single-stranded positive-sense RNA Virus, is a member of the PotexVirus genus of Alphaflexiviridae. It can infect at least 12 species of Bamboo, causing a huge economic impact on the Bamboo industry in Taiwan. The study of BaMV did not start extensively until the completion of the full-length sequencing of genomic RNA of BaMV and generation of the BaMV infectious cDNA clone in the early 1990s. Since then, BaMV has been extensively studied at the molecular, cellular and ecological level, covering both basic and applied researches, by a group of researchers in Taiwan. In this eBook, the content comprises 6 reviews and 4 articles. Seven of them are involved in the infection of BaMV covering viral RNA replication, viral RNA trafficking, and the host factors. Two of them are related to the vector transmission and the ecology of BaMV. The last one is the application of using BaMV as a viral vector to produce vaccines in plants.
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proliferating cell nuclear antigen suppresses rna replication of Bamboo Mosaic Virus through an interaction with the viral genome
Journal of Virology, 2019Co-Authors: Chengcheng Lee, Yauheiu Hsu, Ying-wen Huang, Jhihwei Wang, Weiming Leu, Yuting Huang, Menghsiao MengAbstract:Bamboo Mosaic Virus (BaMV), a member of the PotexVirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail Mosaic Virus and Potato Virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexViruses.IMPORTANCE Knowing the dynamic interplay between plant RNA Viruses and their host is a basic step toward first understanding how the Viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexViruses, including Bamboo Mosaic Virus, Foxtail Mosaic Virus, and Potato Virus X Based on the tests on Bamboo Mosaic Virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the Virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexViruses in the rapidly dividing tissues of plants.
Na-sheng Lin - One of the best experts on this subject based on the ideXlab platform.
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stable display of artificially long foreign antigens on chimeric Bamboo Mosaic Virus particles
Viruses, 2021Co-Authors: Tsunghsien Chen, Chin-wei Lee, Na-sheng Lin, Yumin Feng, Yauheiu HsuAbstract:Plant Viruses can be genetically modified to generate chimeric Virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo Mosaic Virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease Virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete Virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant Virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.
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disrupting the homeostasis of high mobility group protein promotes the systemic movement of Bamboo Mosaic Virus
Frontiers in Plant Science, 2020Co-Authors: Mazen Alazem, Chihhao Chang, Ning Cheng, Na-sheng LinAbstract:Viruses hijack various organelles and machineries for their replication and movement. Ever more lines of evidence indicate that specific nuclear factors are involved in systemic trafficking of several Viruses. However, how such factors regulate viral systemic movement remains unclear. Here, we identify a novel role for Nicotiana benthamiana high mobility group nucleoprotein (NbHMG1/2a) in Virus movement. Although infection of N. benthamiana with Bamboo Mosaic Virus (BaMV) decreased NbHMG1/2a expression levels, nuclear-localized NbHMG1/2a protein was shuttled out of the nucleus into cytoplasm upon BaMV infection. NbHMG1/2a knockdown or even overexpression did not affect BaMV accumulation in inoculated leaves, but it did enhance systemic movement of the Virus. Interestingly, the positive regulator Rap-GTPase activation protein 1 was highly upregulated upon infection with BaMV, whereas the negative regulator thioredoxin h protein was greatly reduced, no matter if NbHMG1a/2a was silenced or overexpressed. Our findings indicate that NbHMG1/2a may have a role in plant defense responses. Once its homeostasis is disrupted, expression of relevant host factors may be perturbed that, in turn, facilitates BaMV systemic movement.
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fusion of a novel native signal peptide enhanced the secretion and solubility of bioactive human interferon gamma glycoproteins in nicotiana benthamiana using the Bamboo Mosaic Virus based expression system
Frontiers in Plant Science, 2020Co-Authors: Minchao Jiang, Na-sheng Lin, Weili Hsu, Tsuiling Hsu, Yauheiu HsuAbstract:Plant Viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo Mosaic Virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of 'Ser-Pro' motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis Virus and Influenza Virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.
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molecular biology of Bamboo Mosaic Virus a type member of the potexVirus genus
2020Co-Authors: Yauheiu Hsu, Chinghsiu Tsai, Na-sheng LinAbstract:The flexible filamentous plant Viruses are responsible for more than half of all agricultural loss worldwide. PotexVirus is one of the two most important flexible filamentous plant Viruses. Bamboo Mosaic Virus (BaMV), a single-stranded positive-sense RNA Virus, is a member of the PotexVirus genus of Alphaflexiviridae. It can infect at least 12 species of Bamboo, causing a huge economic impact on the Bamboo industry in Taiwan. The study of BaMV did not start extensively until the completion of the full-length sequencing of genomic RNA of BaMV and generation of the BaMV infectious cDNA clone in the early 1990s. Since then, BaMV has been extensively studied at the molecular, cellular and ecological level, covering both basic and applied researches, by a group of researchers in Taiwan. In this eBook, the content comprises 6 reviews and 4 articles. Seven of them are involved in the infection of BaMV covering viral RNA replication, viral RNA trafficking, and the host factors. Two of them are related to the vector transmission and the ecology of BaMV. The last one is the application of using BaMV as a viral vector to produce vaccines in plants.
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nicotiana benthamiana argonaute10 plays a pro viral role in Bamboo Mosaic Virus infection
New Phytologist, 2019Co-Authors: Ying-wen Huang, Na-sheng Lin, Chinghsiu Tsai, Yauheiu HsuAbstract:RNA silencing is a major defense mechanism against invading Viruses in plants. Argonaute proteins (AGOs) are the key players in RNA silencing. The number of AGO family members involved varies depending on the plant species and they play distinct or sometimes redundant roles in antiviral defense. By using a Virus-induced gene silencing technique, it was found that Nicotiana benthamiana AGO1 restricted Bamboo Mosaic Virus (BaMV) accumulation, but NbAGO10, the closest paralog of NbAGO1, positively regulated BaMV accumulation. Immunoprecipitation assay revealed BaMV Virus-derived small interfering RNAs (vsiRNAs) in NbAGO10 complexes. Transient overexpression of NbAGO10 increased BaMV RNA accumulation, but with co-expression of NbAGO1, BaMV RNA accumulation was reduced, which suggests that NbAGO10 may have competed with NbAGO1 for sequestering BaMV vsiRNA and prevented the formation of RNA-induced silencing complexes. In addition, overexpression of NbAGO10 decreased BaMV vsiRNA accumulation. A host enzyme, small RNA degrading nuclease 1 (SDN1), also was found to interact with NbAGO10 on in vivo pull-down assay. Silencing of SDN1 elevated BaMV vsiRNA level and decreased BaMV RNA accumulation in N. benthamiana, indicating that NbAGO10 might recruit SDN1 for BaMV vsiRNA degradation. The results herein suggested that NbAGO10 plays a pro-viral role by BaMV vsiRNA sequestration and degradation.
Chinghsiu Tsai - One of the best experts on this subject based on the ideXlab platform.
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dissecting the role of a plant specific rab5 small gtpase nbrabf1 in Bamboo Mosaic Virus infection
Journal of Experimental Botany, 2020Co-Authors: Ying-ping Huang, Chinghsiu Tsai, I-hsuan Chen, Yauhuei Hsu, Peiyu Hou, Chi Ping ChengAbstract:NbRabF1, a small GTPase from Nicotiana benthamiana and a homolog of Arabidopsis thaliana Ara6, plays a key role in regulating Bamboo Mosaic Virus (BaMV) movement by vesicle transport between endosomal membranes. Reducing the expression of NbRabF1 in N. benthamiana by Virus-induced gene silencing decreased the accumulation of BaMV, and with smaller infection foci on inoculated leaves, but had no effect in protoplasts. Furthermore, transient expression of NbRabF1 increased the accumulation of BaMV in inoculated leaves. Thus, NbRabF1 may be involved in the cell-to-cell movement of BaMV. The potential acyl modification sites at the second and third amino acid positions of NbRabF1 were crucial for membrane targeting and BaMV accumulation. The localization of mutant forms of NbRabF1 with the GDP-bound (donor site) and GTP-bound (acceptor site) suggested that NbRabF1 might regulate vesicle trafficking between the Golgi apparatus and plasma membrane. Furthermore, GTPase activity could also be involved in BaMV cell-to-cell movement. Overall, in this study, we identified a small GTPase, NbRabF1, from N. benthamiana that interacts with its activation protein NbRabGAP1 and regulates vesicle transport from the Golgi apparatus to the plasma membrane. We suggest that the BaMV movement complex might move from cell to cell through this vesicle trafficking route.
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the lipid transfer protein 1 from nicotiana benthamiana assists Bamboo Mosaic Virus accumulation
Viruses, 2020Co-Authors: Lingying Chiu, Yauheiu Hsu, I-hsuan Chen, Chinghsiu TsaiAbstract:Host factors play a pivotal role in regulating Virus infection. Uncovering the mechanism of how host factors are involved in Virus infection could pave the way to defeat viral disease. In this study, we characterized a lipid transfer protein, designated NbLTP1 in Nicotiana benthamiana, which was downregulated after Bamboo Mosaic Virus (BaMV) inoculation. BaMV accumulation significantly decreased in NbLTP1-knockdown leaves and protoplasts compared with the controls. The subcellular localization of the NbLTP1-orange fluorescent protein (OFP) was mainly the extracellular matrix. However, when we removed the signal peptide (NbLTP1/ΔSP-OFP), most of the expressed protein targeted chloroplasts. Both NbLTP1-OFP and NbLTP1/ΔSP-OFP were localized in chloroplasts when we removed the cell wall. These results suggest that NbLTP1 may have a secondary targeting signal. Transient overexpression of NbLTP1 had no effect on BaMV accumulation, but that of NbLTP1/ΔSP significantly increased BaMV expression. NbLTP1 may be a positive regulator of BaMV accumulation especially when its expression is associated with chloroplasts, where BaMV replicates. The mutation was introduced to the predicted phosphorylation site to simulate the phosphorylated status, NbLTP/ΔSP/P(+), which could still assist BaMV accumulation. By contrast, a mutant lacking calmodulin-binding or simulates the phosphorylation-negative status could not support BaMV accumulation. The lipid-binding activity of LTP1 was reported to be associated with calmodulin-binding and phosphorylation, by which the C-terminus functional domain of NbLTP1 may play a critical role in BaMV accumulation.
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molecular biology of Bamboo Mosaic Virus a type member of the potexVirus genus
2020Co-Authors: Yauheiu Hsu, Chinghsiu Tsai, Na-sheng LinAbstract:The flexible filamentous plant Viruses are responsible for more than half of all agricultural loss worldwide. PotexVirus is one of the two most important flexible filamentous plant Viruses. Bamboo Mosaic Virus (BaMV), a single-stranded positive-sense RNA Virus, is a member of the PotexVirus genus of Alphaflexiviridae. It can infect at least 12 species of Bamboo, causing a huge economic impact on the Bamboo industry in Taiwan. The study of BaMV did not start extensively until the completion of the full-length sequencing of genomic RNA of BaMV and generation of the BaMV infectious cDNA clone in the early 1990s. Since then, BaMV has been extensively studied at the molecular, cellular and ecological level, covering both basic and applied researches, by a group of researchers in Taiwan. In this eBook, the content comprises 6 reviews and 4 articles. Seven of them are involved in the infection of BaMV covering viral RNA replication, viral RNA trafficking, and the host factors. Two of them are related to the vector transmission and the ecology of BaMV. The last one is the application of using BaMV as a viral vector to produce vaccines in plants.
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nicotiana benthamiana argonaute10 plays a pro viral role in Bamboo Mosaic Virus infection
New Phytologist, 2019Co-Authors: Ying-wen Huang, Na-sheng Lin, Chinghsiu Tsai, Yauheiu HsuAbstract:RNA silencing is a major defense mechanism against invading Viruses in plants. Argonaute proteins (AGOs) are the key players in RNA silencing. The number of AGO family members involved varies depending on the plant species and they play distinct or sometimes redundant roles in antiviral defense. By using a Virus-induced gene silencing technique, it was found that Nicotiana benthamiana AGO1 restricted Bamboo Mosaic Virus (BaMV) accumulation, but NbAGO10, the closest paralog of NbAGO1, positively regulated BaMV accumulation. Immunoprecipitation assay revealed BaMV Virus-derived small interfering RNAs (vsiRNAs) in NbAGO10 complexes. Transient overexpression of NbAGO10 increased BaMV RNA accumulation, but with co-expression of NbAGO1, BaMV RNA accumulation was reduced, which suggests that NbAGO10 may have competed with NbAGO1 for sequestering BaMV vsiRNA and prevented the formation of RNA-induced silencing complexes. In addition, overexpression of NbAGO10 decreased BaMV vsiRNA accumulation. A host enzyme, small RNA degrading nuclease 1 (SDN1), also was found to interact with NbAGO10 on in vivo pull-down assay. Silencing of SDN1 elevated BaMV vsiRNA level and decreased BaMV RNA accumulation in N. benthamiana, indicating that NbAGO10 might recruit SDN1 for BaMV vsiRNA degradation. The results herein suggested that NbAGO10 plays a pro-viral role by BaMV vsiRNA sequestration and degradation.
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autophagy is involved in assisting the replication of Bamboo Mosaic Virus in nicotiana benthamiana
Journal of Experimental Botany, 2019Co-Authors: Ying-ping Huang, Ying-wen Huang, Yauhuei Hsu, Yungjen Hsiao, Chinghsiu TsaiAbstract:Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo Mosaic Virus (BaMV), a single-stranded positive-sense RNA Virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.
Menghsiao Meng - One of the best experts on this subject based on the ideXlab platform.
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proliferating cell nuclear antigen suppresses rna replication of Bamboo Mosaic Virus through an interaction with the viral genome
Journal of Virology, 2019Co-Authors: Chengcheng Lee, Yauheiu Hsu, Ying-wen Huang, Jhihwei Wang, Weiming Leu, Yuting Huang, Menghsiao MengAbstract:Bamboo Mosaic Virus (BaMV), a member of the PotexVirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail Mosaic Virus and Potato Virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexViruses.IMPORTANCE Knowing the dynamic interplay between plant RNA Viruses and their host is a basic step toward first understanding how the Viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexViruses, including Bamboo Mosaic Virus, Foxtail Mosaic Virus, and Potato Virus X Based on the tests on Bamboo Mosaic Virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the Virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexViruses in the rapidly dividing tissues of plants.
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mitogen activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo Mosaic Virus in nicotiana benthamiana
Molecular Plant Pathology, 2018Co-Authors: Chengcheng Lee, Yauheiu Hsu, Yuting Huang, Chiahsin Hsueh, Menghsiao MengAbstract:In plants, the mitogen-activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen-associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo Mosaic Virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV-encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity-dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail Mosaic Virus but decreased Potato Virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad-spectrum resistance through genetically manipulating a single player in MAPK cascades.
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Function and Structural Organization of the Replication Protein of Bamboo Mosaic Virus.
Frontiers in microbiology, 2017Co-Authors: Menghsiao Meng, Chengcheng LeeAbstract:The genus PotexVirus is one of the eight genera belonging to the family Alphaflexiviridae according to the Virus Taxonomy 2015 released by International Committee on Taxonomy of Viruses (www.ictvonline.org/index.asp). Currently, the genus contains 35 known species including many agricultural important Viruses, e.g. Potato Virus X (PVX). Members of this genus are characterized by flexuous, filamentous virions of 13 nm in diameter and 470–580 nm in length. A potexVirus has a monopartite positive-strand RNA genome, encoding five open-reading frames (ORFs), with a cap structure at the 5’ end and a poly(A) tail at the 3’ end. Besides PVX, Bamboo Mosaic Virus (BaMV) is another potexVirus that has received intensive attention due to the wealth of knowledge on the molecular biology of the Virus. In this review, we discuss the enzymatic activities associated with each of the functional domains of the BaMV replication protein, a 155-kDa polypeptide encoded by ORF1. The unique cap formation mechanism, which may be conserved across the alphaVirus superfamily, is particularly addressed. The recently identified interactions between the replication protein and the plant host factors are also described.
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The Stable Association of Virion with the Triple-gene- block Protein 3-based Complex of Bamboo Mosaic Virus
2016Co-Authors: Sheng Lin, Yauheiu Hsu, Menghsiao Meng, Banyang ChangAbstract:The triple-gene-block protein 3 (TGBp3) of Bamboo Mosaic Virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the Virus intracellularly. However, the Virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of Virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in Virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexVirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membran
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promotion of Bamboo Mosaic Virus accumulation in nicotiana benthamiana by 5 3 exonuclease nbxrn4
Frontiers in Microbiology, 2016Co-Authors: Chengcheng Lee, Yauheiu Hsu, Yu-tsung Han, Yuting Huang, Tzuling Lin, Jhewei Lin, Menghsiao MengAbstract:Bamboo Mosaic Virus (BaMV) has a 6.4-kb (+) sense RNA genome with a 5’ cap and a 3’ poly(A) tail. ORF1 of this potexVirus encodes a 155-kDa replication protein responsible for the viral RNA replication/transcription and 5’ cap formation. To learn more about the replication complex of BaMV, a protein preparation enriched in the 155-kDa replication protein was obtained from Nicotiana benthamiana by a protocol involving agroinfiltration and immunoprecipitation. Subsequent analysis by SDS-PAGE and mass spectrometry identified a handful of host proteins that may participate in the viral replication. Among them, the cytoplasmic exoribonuclease NbXRN4 particularly caught our attention. NbXRN4 has been shown to have an antiviral activity against Tomato bushy stunt Virus and Tomato Mosaic Virus. In Arabidopsis, the enzyme could reduce RNAi- and miRNA-mediated RNA decay. This study found that downregulation of NbXRN4 greatly decreased BaMV accumulation, while overexpression of NbXRN4 resulted in an opposite effect. Mutations at the catalytically essential residues abolished the function of NbXRN4 in the increase of BaMV accumulation. Nonetheless, NbXRN4 was still able to promote BaMV accumulation in the presence of the RNA silencing suppressor P19. In summary, the replication efficiency of BaMV may be improved by the exoribonuclease activity of NbXRN4.
Ying-wen Huang - One of the best experts on this subject based on the ideXlab platform.
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proliferating cell nuclear antigen suppresses rna replication of Bamboo Mosaic Virus through an interaction with the viral genome
Journal of Virology, 2019Co-Authors: Chengcheng Lee, Yauheiu Hsu, Ying-wen Huang, Jhihwei Wang, Weiming Leu, Yuting Huang, Menghsiao MengAbstract:Bamboo Mosaic Virus (BaMV), a member of the PotexVirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail Mosaic Virus and Potato Virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexViruses.IMPORTANCE Knowing the dynamic interplay between plant RNA Viruses and their host is a basic step toward first understanding how the Viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexViruses, including Bamboo Mosaic Virus, Foxtail Mosaic Virus, and Potato Virus X Based on the tests on Bamboo Mosaic Virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the Virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexViruses in the rapidly dividing tissues of plants.
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nicotiana benthamiana argonaute10 plays a pro viral role in Bamboo Mosaic Virus infection
New Phytologist, 2019Co-Authors: Ying-wen Huang, Na-sheng Lin, Chinghsiu Tsai, Yauheiu HsuAbstract:RNA silencing is a major defense mechanism against invading Viruses in plants. Argonaute proteins (AGOs) are the key players in RNA silencing. The number of AGO family members involved varies depending on the plant species and they play distinct or sometimes redundant roles in antiviral defense. By using a Virus-induced gene silencing technique, it was found that Nicotiana benthamiana AGO1 restricted Bamboo Mosaic Virus (BaMV) accumulation, but NbAGO10, the closest paralog of NbAGO1, positively regulated BaMV accumulation. Immunoprecipitation assay revealed BaMV Virus-derived small interfering RNAs (vsiRNAs) in NbAGO10 complexes. Transient overexpression of NbAGO10 increased BaMV RNA accumulation, but with co-expression of NbAGO1, BaMV RNA accumulation was reduced, which suggests that NbAGO10 may have competed with NbAGO1 for sequestering BaMV vsiRNA and prevented the formation of RNA-induced silencing complexes. In addition, overexpression of NbAGO10 decreased BaMV vsiRNA accumulation. A host enzyme, small RNA degrading nuclease 1 (SDN1), also was found to interact with NbAGO10 on in vivo pull-down assay. Silencing of SDN1 elevated BaMV vsiRNA level and decreased BaMV RNA accumulation in N. benthamiana, indicating that NbAGO10 might recruit SDN1 for BaMV vsiRNA degradation. The results herein suggested that NbAGO10 plays a pro-viral role by BaMV vsiRNA sequestration and degradation.
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autophagy is involved in assisting the replication of Bamboo Mosaic Virus in nicotiana benthamiana
Journal of Experimental Botany, 2019Co-Authors: Ying-ping Huang, Ying-wen Huang, Yauhuei Hsu, Yungjen Hsiao, Chinghsiu TsaiAbstract:Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo Mosaic Virus (BaMV), a single-stranded positive-sense RNA Virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.
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chloroplast hsp70 isoform is required for age dependent tissue preference of Bamboo Mosaic Virus in mature nicotiana benthamiana leaves
Molecular Plant-microbe Interactions, 2017Co-Authors: Ying-wen Huang, Na-sheng Lin, Chinghsiu Tsai, Yauheiu HsuAbstract:Plant Viruses may exhibit age-dependent tissue preference in their hosts but the underlying mechanisms are not well understood. In this study, we provide several lines of evidence to reveal the determining role of a protein of the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) family, NbcpHsp70-2, involved in the preference of Bamboo Mosaic Virus (BaMV) to infect older tissues. NbcpHsp70 family proteins were identified in complexes pulled down with BaMV replicase as the bait. Among the isoforms of NbcpHsp70, only the specific silencing of NbcpHsp70-2 resulted in the significant decrease of BaMV RNA in N. benthamiana protopalsts, indicating that NbcpHsp70-2 is involved in the efficient replication of BaMV RNA. We further identified the age-dependent import regulation signal contained in the transit peptide of NbcpHsp70-2. Deletion, overexpression, and substitution experiments revealed that the signal in the transit peptide of NbcpHsp70-2 is crucial for both the import of NbcpHsp70-2 into older chloroplasts and the preference of BaMV for infecting older leaves of N. benthamiana. Together, these data demonstrated that BaMV may exploit a cellular age-dependent transportation mechanism to target a suitable environment for viral replication.
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production of japanese encephalitis Virus antigens in plants using Bamboo Mosaic Virus based vector
Frontiers in Microbiology, 2017Co-Authors: Tsunghsien Chen, Jiateh Liao, Na-sheng Lin, Ying-wen Huang, Yiling Lee, Yiling Lin, Yauheiu HsuAbstract:Japanese encephalitis Virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric Virus particle (CVP) strategy based on Bamboo Mosaic Virus (BaMV) for epitope presentation. The chimeric Virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease Virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.
