In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato
RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato...
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| Veröffentlicht in: | Potato research 2020-06, Vol.63 (2), p.143-155 |
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| creator | Sajid, Imtiaz Ahmad Tabassum, Bushra Yousaf, Iqra Khan, Anwar Adeyinka, Olawale Samuel Shahid, Naila Nasir, Idrees Ahmad Husnain, Tayyab |
| description | RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through
Agrobacterium
and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants. |
| doi_str_mv | 10.1007/s11540-019-09433-0 |
| format | Article |
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Agrobacterium
and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.</description><identifier>ISSN: 0014-3065</identifier><identifier>EISSN: 1871-4528</identifier><identifier>DOI: 10.1007/s11540-019-09433-0</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Agriculture ; Antiviral agents ; Biomedical and Life Sciences ; Biotechnology ; Chlorosis ; Coat protein ; Cultivars ; Disease control ; Food ; Gene expression ; Gene sequencing ; Gene silencing ; Genes ; Genetic engineering ; Genetically engineered foods ; Genetically modified plants ; In vivo methods and tests ; Inoculation ; Insertion ; Life Sciences ; MicroRNAs ; miRNA ; Plant Genetics and Genomics ; Plant Sciences ; Plant Systematics/Taxonomy/Biogeography ; Polymerase chain reaction ; Potatoes ; Ribonucleic acid ; RNA ; RNA-mediated interference ; Signs and symptoms ; siRNA ; Stimulators ; Transgenic plants ; Vegetables ; Viruses</subject><ispartof>Potato research, 2020-06, Vol.63 (2), p.143-155</ispartof><rights>European Association for Potato Research 2019</rights><rights>COPYRIGHT 2020 Springer</rights><rights>Potato Research is a copyright of Springer, (2019). All Rights Reserved.</rights><rights>European Association for Potato Research 2019.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c414t-abfcbf8c6c16ca9006e73122ba20c100aa8c86a875201006a6e92958649292733</citedby><cites>FETCH-LOGICAL-c414t-abfcbf8c6c16ca9006e73122ba20c100aa8c86a875201006a6e92958649292733</cites><orcidid>0000-0002-2460-2706</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s11540-019-09433-0$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11540-019-09433-0$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids></links><search><creatorcontrib>Sajid, Imtiaz Ahmad</creatorcontrib><creatorcontrib>Tabassum, Bushra</creatorcontrib><creatorcontrib>Yousaf, Iqra</creatorcontrib><creatorcontrib>Khan, Anwar</creatorcontrib><creatorcontrib>Adeyinka, Olawale Samuel</creatorcontrib><creatorcontrib>Shahid, Naila</creatorcontrib><creatorcontrib>Nasir, Idrees Ahmad</creatorcontrib><creatorcontrib>Husnain, Tayyab</creatorcontrib><title>In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato</title><title>Potato research</title><addtitle>Potato Res</addtitle><description>RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through
Agrobacterium
and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.</description><subject>Agriculture</subject><subject>Antiviral agents</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Chlorosis</subject><subject>Coat protein</subject><subject>Cultivars</subject><subject>Disease control</subject><subject>Food</subject><subject>Gene expression</subject><subject>Gene sequencing</subject><subject>Gene silencing</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genetically engineered foods</subject><subject>Genetically modified plants</subject><subject>In vivo methods and tests</subject><subject>Inoculation</subject><subject>Insertion</subject><subject>Life Sciences</subject><subject>MicroRNAs</subject><subject>miRNA</subject><subject>Plant Genetics and Genomics</subject><subject>Plant Sciences</subject><subject>Plant Systematics/Taxonomy/Biogeography</subject><subject>Polymerase chain reaction</subject><subject>Potatoes</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA-mediated interference</subject><subject>Signs and symptoms</subject><subject>siRNA</subject><subject>Stimulators</subject><subject>Transgenic plants</subject><subject>Vegetables</subject><subject>Viruses</subject><issn>0014-3065</issn><issn>1871-4528</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><recordid>eNp9kV1LBCEUhiUK2j7-QFdC17MdHXWcyyVqW1gq2oruxDFnMGa1dDbYf581QQQRXhzE5zlHfRE6ITAlANVZIoQzKIDUBdSsLAvYQRMiK1IwTuUumgAQVpQg-D46SOkFgHFO-AQ9LTx-dO8Bz623eOV6643zHQ4tvg2DHkI-jZuEn3Czxau17nu88IONrY2ZtPjuepaw8_g-ap8665359o7QXqv7ZI-_6yF6uLy4P78qljfzxflsWRhG2FDopjVNK40wRBhdAwhblYTSRlMw-WlaSyOFlhWnkLdCC1vTmkvBcqFVWR6i07HvawxvG5sG9RI20eeRijLgNfBSsH-p3EVAxUn9Q3W6t8r5NgxRm7VLRs0qQgSvQMpMTf-g8nq2a2eCt23-xd8CHQUTQ0rRtuo1urWOW0VAfcanxvhUjk99xacgS-UopQz7zsafG_9jfQC6KZhH</recordid><startdate>20200601</startdate><enddate>20200601</enddate><creator>Sajid, Imtiaz Ahmad</creator><creator>Tabassum, Bushra</creator><creator>Yousaf, Iqra</creator><creator>Khan, Anwar</creator><creator>Adeyinka, Olawale Samuel</creator><creator>Shahid, Naila</creator><creator>Nasir, Idrees Ahmad</creator><creator>Husnain, Tayyab</creator><general>Springer Netherlands</general><general>Springer</general><general>Springer Nature B.V</general><scope>AAYXX</scope><scope>CITATION</scope><orcidid>https://orcid.org/0000-0002-2460-2706</orcidid></search><sort><creationdate>20200601</creationdate><title>In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato</title><author>Sajid, Imtiaz Ahmad ; Tabassum, Bushra ; Yousaf, Iqra ; Khan, Anwar ; Adeyinka, Olawale Samuel ; Shahid, Naila ; Nasir, Idrees Ahmad ; Husnain, Tayyab</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c414t-abfcbf8c6c16ca9006e73122ba20c100aa8c86a875201006a6e92958649292733</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Agriculture</topic><topic>Antiviral agents</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnology</topic><topic>Chlorosis</topic><topic>Coat protein</topic><topic>Cultivars</topic><topic>Disease control</topic><topic>Food</topic><topic>Gene expression</topic><topic>Gene sequencing</topic><topic>Gene silencing</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Genetically engineered foods</topic><topic>Genetically modified plants</topic><topic>In vivo methods and tests</topic><topic>Inoculation</topic><topic>Insertion</topic><topic>Life Sciences</topic><topic>MicroRNAs</topic><topic>miRNA</topic><topic>Plant Genetics and Genomics</topic><topic>Plant Sciences</topic><topic>Plant Systematics/Taxonomy/Biogeography</topic><topic>Polymerase chain reaction</topic><topic>Potatoes</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA-mediated interference</topic><topic>Signs and symptoms</topic><topic>siRNA</topic><topic>Stimulators</topic><topic>Transgenic plants</topic><topic>Vegetables</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sajid, Imtiaz Ahmad</creatorcontrib><creatorcontrib>Tabassum, Bushra</creatorcontrib><creatorcontrib>Yousaf, Iqra</creatorcontrib><creatorcontrib>Khan, Anwar</creatorcontrib><creatorcontrib>Adeyinka, Olawale Samuel</creatorcontrib><creatorcontrib>Shahid, Naila</creatorcontrib><creatorcontrib>Nasir, Idrees Ahmad</creatorcontrib><creatorcontrib>Husnain, Tayyab</creatorcontrib><collection>CrossRef</collection><jtitle>Potato research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sajid, Imtiaz Ahmad</au><au>Tabassum, Bushra</au><au>Yousaf, Iqra</au><au>Khan, Anwar</au><au>Adeyinka, Olawale Samuel</au><au>Shahid, Naila</au><au>Nasir, Idrees Ahmad</au><au>Husnain, Tayyab</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato</atitle><jtitle>Potato research</jtitle><stitle>Potato Res</stitle><date>2020-06-01</date><risdate>2020</risdate><volume>63</volume><issue>2</issue><spage>143</spage><epage>155</epage><pages>143-155</pages><issn>0014-3065</issn><eissn>1871-4528</eissn><abstract>RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through
Agrobacterium
and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><doi>10.1007/s11540-019-09433-0</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-2460-2706</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Agriculture Antiviral agents Biomedical and Life Sciences Biotechnology Chlorosis Coat protein Cultivars Disease control Food Gene expression Gene sequencing Gene silencing Genes Genetic engineering Genetically engineered foods Genetically modified plants In vivo methods and tests Inoculation Insertion Life Sciences MicroRNAs miRNA Plant Genetics and Genomics Plant Sciences Plant Systematics/Taxonomy/Biogeography Polymerase chain reaction Potatoes Ribonucleic acid RNA RNA-mediated interference Signs and symptoms siRNA Stimulators Transgenic plants Vegetables Viruses |
| title | In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato |
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