Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses
Publication Date
December 22, 2015
Journal
PLOS Biology
Authors
Nahid Shamandi, Matthias Zytnicki, Cyril Charbonnel, Emilie Elvira Matelot, et al
Volume
13
Issue
12
Pages
e1002326
DOI
https://dx.plos.org/10.1371/journal.pbio.1002326
Publisher URL
http://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.1002326
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/26696443
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687873
Europe PMC
http://europepmc.org/abstract/MED/26696443
Web of Science
000368443000018
Scopus
84953207767
Mendeley
http://www.mendeley.com/research/plants-encode-general-sirna-suppressor-induced-suppressed-viruses
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Mendeley | Further Information

{"title"=>"Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses", "type"=>"journal", "authors"=>[{"first_name"=>"Nahid", "last_name"=>"Shamandi", "scopus_author_id"=>"57039180000"}, {"first_name"=>"Matthias", "last_name"=>"Zytnicki", "scopus_author_id"=>"15064918100"}, {"first_name"=>"Cyril", "last_name"=>"Charbonnel", "scopus_author_id"=>"36611378500"}, {"first_name"=>"Emilie", "last_name"=>"Elvira-Matelot", "scopus_author_id"=>"25922406800"}, {"first_name"=>"Aurore", "last_name"=>"Bochnakian", "scopus_author_id"=>"57039181700"}, {"first_name"=>"Pascale", "last_name"=>"Comella", "scopus_author_id"=>"7005685606"}, {"first_name"=>"Allison C.", "last_name"=>"Mallory", "scopus_author_id"=>"7004401197"}, {"first_name"=>"Gersende", "last_name"=>"Lepère", "scopus_author_id"=>"24341314000"}, {"first_name"=>"Julio", "last_name"=>"Sáez-Vásquez", "scopus_author_id"=>"6602944712"}, {"first_name"=>"Hervé", "last_name"=>"Vaucheret", "scopus_author_id"=>"7004443166"}], "year"=>2015, "source"=>"PLoS Biology", "identifiers"=>{"issn"=>"15457885", "sgr"=>"84953207767", "doi"=>"10.1371/journal.pbio.1002326", "scopus"=>"2-s2.0-84953207767", "isbn"=>"1869-1889 (Electronic)\\r1674-7305 (Linking)", "pmid"=>"26696443", "pui"=>"607461882"}, "id"=>"5e52bf12-de7f-3f21-8e73-7be3e3ad041c", "abstract"=>"Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.", "link"=>"http://www.mendeley.com/research/plants-encode-general-sirna-suppressor-induced-suppressed-viruses", "reader_count"=>110, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>23, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>34, "Student > Postgraduate"=>2, "Student > Master"=>16, "Other"=>4, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>8}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>23, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>34, "Student > Postgraduate"=>2, "Student > Master"=>16, "Other"=>4, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>8}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>10, "Agricultural and Biological Sciences"=>95, "Medicine and Dentistry"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>95}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"Argentina"=>1, "Austria"=>1, "United States"=>2, "Philippines"=>1, "Brazil"=>2, "United Kingdom"=>2, "Slovenia"=>1, "Chile"=>1}, "group_count"=>6}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2615334"], "description"=>"<p><b>A</b>) Pictures of water (mock)-, TCV-, TVCV-, CMV-, and TYMV-inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2. Ten-day-old plants were inoculated. Pictures were taken three weeks following inoculation. <b>B</b>) RNA gel blot detection of TCV, TVCV, CMV, and TYMV RNAs and siRNAs in total aerial parts of mock- and virus-inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2. <i>U6</i> was used as a loading control. The <i>U6</i> panels for CMV and TVCV are similar to those in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.g007\" target=\"_blank\">Fig 7A</a> because the blots used in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.g007\" target=\"_blank\">Fig 7A</a> were stripped and rehybridized with CMV and TVCV probes to produce Fig 8B.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629312, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g008", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Impact_of_RTL1_on_virus_infection_/1629312", "title"=>"Impact of RTL1 on virus infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615335", "https://ndownloader.figshare.com/files/2615336", "https://ndownloader.figshare.com/files/2615337", "https://ndownloader.figshare.com/files/2615338", "https://ndownloader.figshare.com/files/2615339", "https://ndownloader.figshare.com/files/2615340", "https://ndownloader.figshare.com/files/2615341", "https://ndownloader.figshare.com/files/2615342", "https://ndownloader.figshare.com/files/2615343", "https://ndownloader.figshare.com/files/2615344", "https://ndownloader.figshare.com/files/2615345", "https://ndownloader.figshare.com/files/2615346", "https://ndownloader.figshare.com/files/2615347", "https://ndownloader.figshare.com/files/2615348", "https://ndownloader.figshare.com/files/2615349", "https://ndownloader.figshare.com/files/2615350", "https://ndownloader.figshare.com/files/2615351", "https://ndownloader.figshare.com/files/2615352"], "description"=>"<div><p>Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces <i>RTL1</i> mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.</p></div>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629313, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.1002326.s001", "https://dx.doi.org/10.1371/journal.pbio.1002326.s002", "https://dx.doi.org/10.1371/journal.pbio.1002326.s003", "https://dx.doi.org/10.1371/journal.pbio.1002326.s004", "https://dx.doi.org/10.1371/journal.pbio.1002326.s005", "https://dx.doi.org/10.1371/journal.pbio.1002326.s006", "https://dx.doi.org/10.1371/journal.pbio.1002326.s007", "https://dx.doi.org/10.1371/journal.pbio.1002326.s008", "https://dx.doi.org/10.1371/journal.pbio.1002326.s009", "https://dx.doi.org/10.1371/journal.pbio.1002326.s010", "https://dx.doi.org/10.1371/journal.pbio.1002326.s011", "https://dx.doi.org/10.1371/journal.pbio.1002326.s012", "https://dx.doi.org/10.1371/journal.pbio.1002326.s013", "https://dx.doi.org/10.1371/journal.pbio.1002326.s014", "https://dx.doi.org/10.1371/journal.pbio.1002326.s015", "https://dx.doi.org/10.1371/journal.pbio.1002326.s016", "https://dx.doi.org/10.1371/journal.pbio.1002326.s017", "https://dx.doi.org/10.1371/journal.pbio.1002326.s018"], "stats"=>{"downloads"=>5, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Plants_Encode_a_General_siRNA_Suppressor_That_Is_Induced_and_Suppressed_by_Viruses_/1629313", "title"=>"Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2016-01-05 14:53:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615327"], "description"=>"<p><b>A)</b> RNA extracted from the total aerial part of wild-type plants (Col) three weeks after inoculation with water (mock), TCV, TVCV, CMV, or TYMV were subjected to oligo-dT reverse transcription followed by qPCR with primers specific to <i>RTL1</i>, <i>RTL2</i>, or <i>RTL3</i>. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>B)</b> RNA extracted from leaves and roots of 3 wk-old wild-type plants (Col) were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>C)</b> RNA extracted from leaves of 3 wk-old wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag#2</i> (<i>RTL1-Flag</i> #2) plants were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>D</b>) Onion epidermial cells transformed with a <i>35S</i>:<i>RTL1-GFP</i> construct were imaged using a Zeiss Axioskop 2 microscope and recorded using a Leica DC 300 FX digital camera (Leica). <b>E</b>) Proteins were extracted from 18-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants and hybridized with an anti-RTL1 antibody. Hybridization with an anti-RPL13 antibody serves as a loading control. <b>F)</b> Immunostaining of root cells from 8-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants was performed using an anti-RTL1 antibody and revealed with Alexa 488.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629305, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g001", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RTL1_expression_and_localization_/1629305", "title"=>"RTL1 expression and localization.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615328"], "description"=>"<p><b>A</b>) Range of phenotypes of transgenic plants overexpressing RTL1. A wild-type plant (Col) is shown as control. <b>B</b>) and <b>C</b>) Gel blots of total RNA from flowers of wild-type (Col), <i>35S</i>:<i>RTL1</i> (<i>RTL1</i>) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants were hybridized with the indicated probes. For B, a single blot was successively hybridized, stripped, and rehybridized with the different probes. For C, ten identical blots were hybridized each with a different probe and then rehybridized with U6 as a loading control. A representative U6 control is shown. All U6 controls can be seen in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.s002\" target=\"_blank\">S1 Fig</a>.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629306, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phenotype_of_35S_RTL1_plants_and_RNA_gel_blot_analysis_of_small_RNAs_/1629306", "title"=>"Phenotype of <i>35S</i>:<i>RTL1</i> plants and RNA gel blot analysis of small RNAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615329"], "description"=>"<p>Small RNAs from flowers of wild-type (Col), <i>dcl2dcl3dcl4 (dcl234)</i> mutant and the <i>35S</i>:<i>RTL1 (RTL1)</i> plant #1 analyzed in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.g002\" target=\"_blank\">Fig 2</a> were subjected to high throughput sequencing. <b>A</b>) Size distribution of reads that perfectly match the <i>Arabidopsis</i> nuclear genome, excluding rRNA and tRNA. The proportion of each size of small RNA is indicated by a different color: 21 nt (blue), 22 nt (green), 23 nt (pink) and 24 nt (red) and a gradient of grey for 17 to 20 nt and 25 to 30 nt. <b>B</b>) Normalized abundance of siRNAs spanning the nuclear genome. Only siRNAs matching a unique genomic location without ambiguity were considered. Normalization was made to the total of conserved miRNAs.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629307, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g003", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Plants_overexpressing_RTL1_have_reduced_global_levels_of_siRNAs_/1629307", "title"=>"Plants overexpressing RTL1 have reduced global levels of siRNAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615330"], "description"=>"<p><b>A</b>) ta-siRNAs. <b>B</b>) endoIR-siRNas. <b>C</b>) PolIV/PolV-siRNAs. Small RNAs from wild-type (Col), <i>35S</i>:<i>RTL1</i> (<i>RTL1</i>) transgenic plants and <i>dcl2dcl3dcl4 (dcl234)</i> mutants were classified as ta-siRNAs, endoIR-siRNas, or PolIV/PolV-siRNAs based on published annotation. Small RNA abundance was normalized to the total amount of conserved miRNAs. Each size of small RNA is indicated by a different color: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629308, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g004", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Normalized_abundance_of_the_three_major_classes_of_endogenous_siRNAs_/1629308", "title"=>"Normalized abundance of the three major classes of endogenous siRNAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615331"], "description"=>"<p><b>A</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. Low molecular weight (LMW) RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>B</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) and a <i>35S</i>:<i>GUS</i> construct (<i>GUS)</i> together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with U6 as loading control. High molecular weight RNAs were hybridized with a <i>GUS</i> probe and with 25S as loading control. <b>C</b>) The <i>Arabidopsis</i> line <i>L1</i> carrying a <i>35S</i>:<i>GUS</i> transgene silenced by PTGS was transformed with a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>. LMW <i>GUS</i> RNAs from two independent transformants were analyzed. Note that the images presented in this panel are internal to the images presented in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.s010\" target=\"_blank\">S9C Fig</a>. <b>D</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, tagged constructs (<i>RTL1-Myc</i>, <i>Myc-RTL1</i>, <i>RTL1-Flag</i>, <i>Flag-RTL1</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>E</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type tagged <i>35S</i>:<i>RTL1-Myc</i> construct <i>(RTL1-Myc)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. Proteins were extracted and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>F</b>) The <i>Arabidopsis</i> line <i>L1</i> was transformed with either a wild-type-tagged <i>35S</i>:<i>RTL1</i> construct <i>(RTL1-Myc)</i> or a tagged construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>). Proteins were extracted from three independent <i>RTL1-Myc</i> transformants and eight independent <i>RTL1mR3-Myc</i> transformants and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>G</b>) LMW RNAs from <i>RTL1-Myc</i> and <i>RTL1mR3-Myc</i> transformants expressing comparable amount of proteins were hybridized with <i>GUS</i> and <i>TAS2</i> probes and with <i>U6</i> as a loading control.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629309, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g005", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_RTL1_RNaseIII_domain_is_required_for_inhibition_of_transgene_PTGS_/1629309", "title"=>"The RTL1 RNaseIII domain is required for inhibition of transgene PTGS.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615332"], "description"=>"<p><b>A</b>) RNA gel blot detection of <i>IR71</i> precursor RNA in wild-type (Col), Col transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>Col/RTL1</i>), <i>dcl234</i>, and <i>dcl234</i> transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>dcl234/RTL1</i>). Transformants exhibiting a strong RTL1 developmental phenotype were analyzed. High molecular weight (HMW) RNAs extracted from flowers were hybridized with a probe complementary to the <i>IR71</i> RNA and with <i>25S</i> as loading control. <b>B</b>) RNAs extracted from wild-type seedlings were incubated or not with wild-type His-RTL1 and subjected to RT-PCR to detect IR71, IR2039, and At3g18145 (3’UTR) precursor RNAs. <b>C</b>) RNAs extracted from wild-type seedlings were incubated with wild-type or mutant His-RTL1 and subjected to RT-PCR reactions to detect At3g18145 (3’UTR) precursor RNAs. Comassie blue-stained gel shows approximately 200 ng of wild-type and mutant proteins. A schematic representation of RTL1 (residues 1–289) is shown at the top. Black and grey boxes correspond to RNaseIII and dsRBD (double stranded RNA Binding Domain) motifs, respectively. The conserved amino acids in the RNase III signature motif are highlighted in black and the residues E86, E89, E92, and D96 mutated in recombinant proteins indicated by an asterisk. <b>D</b>) RNAs extracted from wild-type seedlings were denaturated or not before incubation with His-RTL1 and subjected to RT-PCR reactions to detect IR71 precursor RNAs. RT-PCR amplification of U3 snoRNA sequences shows similar amount of RNA in each reaction.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629310, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g006", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RTL1_cleaves_dsRNA_/1629310", "title"=>"RTL1 cleaves dsRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-05 14:53:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/2615333"], "description"=>"<p><b>A</b>) RNAs extracted from water (mock)- or virus inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2 were hybridized with a <i>TAS2</i> probe and <i>U6</i> as a loading control. <b>B</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>, a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a <i>35S</i>:<i>VSR</i> construct (<i>P38</i>, <i>P38GA2</i>, <i>P1/HC-Pro</i>, <i>2b</i>, <i>or P69</i>), or a mix of the <i>35S</i>:<i>RTL1</i> and <i>35S</i>:<i>VSR</i> constructs. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control.</p>", "links"=>[], "tags"=>["dcl", "General siRNA Suppressor", "RTL 1 activity", "RTL 1 cleavage", "virus infection", "RTL 1", "RTL 1 expression", "RTL 1 mRNA accumulation", "plant siRNA pathways", "rnase"], "article_id"=>1629311, "categories"=>["Biological Sciences"], "users"=>["Nahid Shamandi", "Matthias Zytnicki", "Cyril Charbonnel", "Emilie Elvira-Matelot", "Aurore Bochnakian", "Pascale Comella", "Allison C. Mallory", "Gersende Lepère", "Julio Sáez-Vásquez", "Hervé Vaucheret"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002326.g007", "stats"=>{"downloads"=>2, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RTL1_activity_is_suppressed_by_VSRs_/1629311", "title"=>"RTL1 activity is suppressed by VSRs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2016-01-06 09:32:45"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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