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Kirankumar S Mysore - One of the best experts on this subject based on the ideXlab platform.
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tobacco rattle virus based virus induced gene silencing in Nicotiana benthamiana
Nature Protocols, 2014Co-Authors: Muthappa Senthilkumar, Kirankumar S MysoreAbstract:Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2-3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.
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Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana
Nature protocols, 2014Co-Authors: Muthappa Senthil-kumar, Kirankumar S MysoreAbstract:Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2-3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.
Paulo Mazzafera - One of the best experts on this subject based on the ideXlab platform.
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The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.
Plant molecular biology, 2020Co-Authors: Raquel Figueiredo, Juan Pablo Portilla Llerena, Eduardo Kiyota, Sávio Siqueira Ferreira, Bárbara Rocha Cardeli, Sarah Caroline Ribeiro De Souza, Michael Dos Santos Brito, Ladaslav Sodek, Igor Cesarino, Paulo MazzaferaAbstract:A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.
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The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana
Plant Molecular Biology, 2020Co-Authors: Raquel Figueiredo, Juan Pablo Portilla Llerena, Eduardo Kiyota, Sávio Siqueira Ferreira, Bárbara Rocha Cardeli, Sarah Caroline Ribeiro De Souza, Ladaslav Sodek, Igor Cesarino, Michael Santos Brito, Paulo MazzaferaAbstract:Key message A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Abstract Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium -mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β -ketoacyl-CoA synthase ( ShKCS20 ) and caffeic acid- O -methyltransferase ( ShCOMT ). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.
Koen Geuten - One of the best experts on this subject based on the ideXlab platform.
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TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms
BMC Plant Biology, 2018Co-Authors: Heleen Coenen, Tom Viaene, Michiel Vandenbussche, Koen GeutenAbstract:Background: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. Results: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. Conclusions: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.
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TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.
BMC plant biology, 2018Co-Authors: Heleen Coenen, Tom Viaene, Michiel Vandenbussche, Koen GeutenAbstract:MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.
Yule Liu - One of the best experts on this subject based on the ideXlab platform.
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Plant protein P3IP participates in the regulation of autophagy in Nicotiana benthamiana.
Plant signaling & behavior, 2020Co-Authors: Liangliang Jiang, Yule Liu, Xiying Zheng, Jianping Chen, Fei YanAbstract:Autophagy, a bulk degradation system conserved among most eukaryotes, is also involved in responses to viral infection in plant. In our previous study, a new host factor P3IP was identified to interact with RSV (rice stripe virus) p3 and mediate its autophagic degradation to limit the viral infection. Here, we further discovered that P3IP of Nicotiana benthamiana (NbP3IP) participated in regulation of autophagy. Overexpression of NbP3IP induced autophagy and down-regulation of NbP3IP reduced autophagy. Combined the functions of autophagy-mediated plant defense against plant virus and regulation autophagy, we indicate that P3IP participates in the regulation of autophagy.
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Virus-induced gene silencing using artificial miRNAs in Nicotiana benthamiana.
Methods in molecular biology (Clifton N.J.), 2013Co-Authors: Yang Tang, Yizhen Lai, Yule LiuAbstract:Virus-induced gene silencing using artificial microRNAs (MIR VIGS) is a newly developed technique for plant reverse genetic studies. Traditional virus-induced gene silencing (VIGS) assays introduce a large gene fragment, which is expressed and then converted into small RNAs by the endogenous siRNA-based gene silencing machinery of the plant host. By contrast, MIR VIGS uses well-designed miRNAs to induce RNA-mediated silencing of the target gene. Using a single artificial miRNA can provide greater specificity by reducing off-target effects. Here, we describe a detailed protocol for MIR VIGS in Nicotiana benthamiana using a modified Cabbage leaf curl virus (CaLCuV)-based vector.
Raquel Figueiredo - One of the best experts on this subject based on the ideXlab platform.
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The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.
Plant molecular biology, 2020Co-Authors: Raquel Figueiredo, Juan Pablo Portilla Llerena, Eduardo Kiyota, Sávio Siqueira Ferreira, Bárbara Rocha Cardeli, Sarah Caroline Ribeiro De Souza, Michael Dos Santos Brito, Ladaslav Sodek, Igor Cesarino, Paulo MazzaferaAbstract:A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.
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The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana
Plant Molecular Biology, 2020Co-Authors: Raquel Figueiredo, Juan Pablo Portilla Llerena, Eduardo Kiyota, Sávio Siqueira Ferreira, Bárbara Rocha Cardeli, Sarah Caroline Ribeiro De Souza, Ladaslav Sodek, Igor Cesarino, Michael Santos Brito, Paulo MazzaferaAbstract:Key message A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Abstract Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium -mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β -ketoacyl-CoA synthase ( ShKCS20 ) and caffeic acid- O -methyltransferase ( ShCOMT ). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.