Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants

Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants Kheta Ram Tak, Anitha Peter, Deepika Vidyashankar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3567612/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Papaya ringspot virus (PRSV) is the most destructive disease of papaya which limits its production and cultivation worldwide. Pathogen-derived resistance (PDR) approach as a popular strategy has been utilized previously to develop resistant cultivars in papaya. However, the pathogen has already overcome the resistance which necessitates an alternative strategy for PRSV disease management. RNAi is an effective method of obtaining resistant transgenic plants against viruses by inducing RNA silencing via expressing virus-derived double-stranded RNA in plants. The current study deals with the isolation and characterization of the HC-Pro gene from PRSV, the development of intron hairpin RNA construct followed by transformation and confirmation in the T1 generation of Nicotiana benthamiana . The partial coding region of the helper component ( HC-Pro ) gene of PRSV was used to design hairpin RNA which includes a spliceosomal intron inserted between the hairpin RNA arms (HC-Pro ihpRNA). Designed HC-Pro ihpRNA was introduced into the plant expression vector (pBI121) and the recombinant plasmids were transformed into Agrobacterium tumefaciens (LBA4404) followed by transformation into Nicotiana bennthamiana as a model plant. Gene expression analysis of transgenic T1 plants infected with PRSV showed reduced Nb POD, Nb APX and Nb CAT compared to inoculated control plants. Results revealed that the suppression of the HC-Pro gene has an effect similar to that of non-infected control plants and hence confers resistance to PRSV infection. We have demonstrated that transgenic tobacco plants expressing partial PRSV HC-pro gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to PRSV infection. Papaya ringspot virus ihpRNA HC-Pro Nicotiana benthamiana SiRNA Full Text Additional Declarations No competing interests reported. Supplementary Files KhetaramTRANSGENICRESEARCHSUPPLEMENTARYDATA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3567612","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":247460354,"identity":"ad3d9d2b-4fca-4c26-8790-32b600f383a8","order_by":0,"name":"Kheta Ram Tak","email":"","orcid":"","institution":"University of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kheta","middleName":"Ram","lastName":"Tak","suffix":""},{"id":247460355,"identity":"0b0026c8-9c5e-4bb3-8c54-758b21a82218","order_by":1,"name":"Anitha Peter","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYHACNgaGAwxyDAw8UD4zkVqMSdeS2ADXQgjwz25+9pjnzL30DcfPHnzwgcFOjoGd9wBeLRJ3jpkb89wozt1wJi/ZcAZDsjEDM18Cfmtu5LBJ83xIyN1wIMdMmofhQGIDM48BXh3yUC3pBuffEKnFAKzlRkICkEGkFsM7x8wk55xJMJx5442x4QyDZGM2Qlrkbjc/k3hzLEGe73yO4YMPFXZy/Pxn8GthkGBgYALFiMIBsDvB0UQAALUw/gDS8g0ElY6CUTAKRsFIBQCqN0G61v2mdAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Anitha","middleName":"","lastName":"Peter","suffix":""},{"id":247460356,"identity":"ee426eb7-c67f-484f-a0b2-ab012e859cf5","order_by":2,"name":"Deepika Vidyashankar","email":"","orcid":"","institution":"University of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Deepika","middleName":"","lastName":"Vidyashankar","suffix":""}],"badges":[],"createdAt":"2023-11-06 10:29:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3567612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3567612/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47370257,"identity":"a09c32d3-9b02-4659-b8d7-19968736e247","added_by":"auto","created_at":"2023-11-30 13:22:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1303438,"visible":true,"origin":"","legend":"","description":"","filename":"KhetaramTRANSGENICRESEARCHFULLPAPER.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3567612/v1_covered_5d25ffc3-f657-4060-b224-f584da820682.pdf"},{"id":46220430,"identity":"902b54c4-d6a5-45eb-9457-74ce53f9704c","added_by":"auto","created_at":"2023-11-10 12:24:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11113694,"visible":true,"origin":"","legend":"","description":"","filename":"KhetaramTRANSGENICRESEARCHSUPPLEMENTARYDATA.docx","url":"https://assets-eu.researchsquare.com/files/rs-3567612/v1/66b7ff83d2e1412b751d64e4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Papaya ringspot virus, ihpRNA, HC-Pro, Nicotiana benthamiana, SiRNA","lastPublishedDoi":"10.21203/rs.3.rs-3567612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3567612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePapaya ringspot virus\u003c/em\u003e (PRSV) is the most destructive disease of papaya which limits its production and cultivation worldwide. Pathogen-derived resistance (PDR) approach as a popular strategy has been utilized previously to develop resistant cultivars in papaya. However, the pathogen has already overcome the resistance which necessitates an alternative strategy for PRSV disease management. RNAi is an effective method of obtaining resistant transgenic plants against viruses by inducing RNA silencing via expressing virus-derived double-stranded RNA in plants. The current study deals with the isolation and characterization of the HC-Pro gene from PRSV, the development of intron hairpin RNA construct followed by transformation and confirmation in the T1 generation of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. The partial coding region of the helper component (\u003cem\u003eHC-Pro\u003c/em\u003e) gene of PRSV was used to design hairpin RNA which includes a spliceosomal intron inserted between the hairpin RNA arms (HC-Pro ihpRNA). Designed HC-Pro ihpRNA was introduced into the plant expression vector (pBI121) and the recombinant plasmids were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (LBA4404) followed by transformation into \u003cem\u003eNicotiana bennthamiana\u003c/em\u003e as a model plant. Gene expression analysis of transgenic T1 plants infected with PRSV showed reduced \u003cem\u003eNb\u003c/em\u003ePOD, \u003cem\u003eNb\u003c/em\u003eAPX and \u003cem\u003eNb\u003c/em\u003eCAT compared to inoculated control plants. Results revealed that the suppression of the \u003cem\u003eHC-Pro\u003c/em\u003e gene has an effect similar to that of non-infected control plants and hence confers resistance to PRSV infection. We have demonstrated that transgenic tobacco plants expressing partial PRSV HC-pro gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to PRSV infection.\u003c/p\u003e","manuscriptTitle":"Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-10 12:24:27","doi":"10.21203/rs.3.rs-3567612/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1aa18845-da60-4461-8f52-bab636635530","owner":[],"postedDate":"November 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-06T13:59:23+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-10 12:24:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3567612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3567612","identity":"rs-3567612","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}