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Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri
Thesis (MScAgric)--Stellenbosch University, 2022.
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867614111104237568 |
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| access_status_str | Open Access |
| author | De Nicola, Ella Hannan Maria |
| author2 | Maree, H. J. |
| author_browse | De Nicola, Ella Hannan Maria Maree, H. J. |
| author_facet | Maree, H. J. De Nicola, Ella Hannan Maria |
| author_sort | De Nicola, Ella Hannan Maria |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/124583 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:50.651Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/124583 Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri De Nicola, Ella Hannan Maria Maree, H. J. Bester, Rachelle Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology. Genetic vectors Biotechnology Citrus tristeza disease -- South Africa Citrus greening disease -- Control Citrus tristeza virus (CTV) -- Genetic aspects Citrus -- Diseases and pests -- Genetic aspects Small interfering RNA Gene silencing UCTD Thesis (MScAgric)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Citrus greening is an incurable and devastating bacterial disease caused by species of phloem- limited Candidatus Liberibacters, transmitted by two phloem-sap feeding insects, Diaphorina citri and Trioza erytreae. Citrus greening poses a constant threat to the success and stability of citriculture worldwide. The pathogen-insect combination of Candidatus Liberibacter asiaticus (CLas) and D. citri, responsible for the most severe form of citrus greening, Huanglongbing (HLB), was reported in Northern Africa for the first time in 2010 and 2016 respectively. Confirmation that the endemic insect vector of Candidatus Liberibacter africanus (CLaf), T. erytreae, can also transmit CLas has significantly increased the risk of HLB reaching South Africa. Until a cure is discovered, the development of a reliable insect vector control strategy is critical. This study aimed to convert an infectious clone of citrus tristeza virus (CTVfl6) into a virus-induced gene silencing (VIGS) vector that targets critical genes in the citrus greening insect vectors and bioinformatically profile the virus- induced small interfering RNAs (vsiRNAs) produced in planta. This putative control strategy is uniquely specific due to the sequence specificity of the RNAi machinery and the phloem-limited infection of CTV. Four candidate genes, arginine kinase, abnormal wing disc, chitin synthase and ran GTPase, were selected as RNAi targets, and six insect- and gene-specific silencing inserts were constructed. A set of 24 intermediate plasmids was assembled, harbouring the silencing inserts in the 3’ terminal end of CTVfl6. These intermediates differed by silencing insert orientation and presence or absence of an upstream duplicated CTV coat protein controller element to evaluate the influence of CTV VIGS vector configuration on vsiRNA production in planta. Assembly of the complete CTVfl6 VIGS vectors was attempted by replacing the 3’ end of the original sequence with the newly engineered intermediate 3’ end. Only three of the 24 were successfully assembled, two T. erytreae insect-specific and one abnormal wing disc gene-specific. Systemic CTV VIGS vector infection was observed in 22% to 33% of N. benthamiana plants 5-6 weeks post-Agrobacterium tumefaciens-infiltration, indicating that CTVfl6 can tolerate large exogenous insertions in the 3’ terminal end. Inoculation into Mexican lime was attempted for the T. erytreae specific CTV VIGS vectors. As CTV was not yet detectable in Mexican lime at the time of sampling, small RNA (sRNA) sequencing was only conducted on CTV positive N. benthamiana material. The vsiRNA coverage across the CTV VIGS vector genomes was over >99%. Hotspots in vsiRNA alignment depth were observed at two of CTV’s silencing suppressors, providing detail on host plant-virus interaction. Insert-specific vsiRNAs were produced in planta, covering >99.78% of the silencing insert. The production of insect-specific vsiRNAs provides some proof of concept for this vsiRNA delivery strategy. The influence of RNAi-mediated knockdown on insect development and mortality should be tested using greenhouse trials. This study has generated the first putative RNAi-based control strategy for the endemic South African citrus greening vector. This strategy is applicable to mitigating the spread of CLaf and, in the event of CLas reaching South Africa, HLB in an African setting. AFRIKAANSE OPSOMMING: Sitrusvergroeningssiekte is ‘n ongeneesbare en vernietigende bakteriële siekte wat veroorsaak word deur spesies van floeëmbeperkte Candidatus Liberibacters. Die bakterieë word oorgedra deur twee insekte, Diaphorina citri en Trioza erytreae, waneer hulle op floeëm sap voed. Sitrusvergroeningssiekte is ‘n voortdurende bedreiging vir die sukses en stabiliteit van sitrusverbouing regoor die wêreld. Die insek-patogeen kombinasie van D. citri en Candidatus Liberibacter asiaticus (CLas), wat verantwoordelik is vir die ernstigste tipe sitrusvergroening, Huanglongbing (HLB), is onderskeidelik in 2016 en 2010 vir die eerste keer in Noord-Amerika gewaar. Die risiko dat HLB Suid-Afrika bereik is drasties verhoog toe daar bevestig is dat T. erytreae, die endemiese insek vektor van Candidatus Liberibacter africaus (CLaf), ook CLas kan oordra. Totdat daar ‘n behandeling vir HLB gevind word, is dit uiters belangrik dat ‘n betroubare strategie ontwikkel word om die insek vektor te beheer. Hierdie studie het gepoog om ‘n infektiewe kloon van die citrus tristeza virus (CTVfl6) te omskep in ‘n virus-geïnduseerde geenonderdrukking (VIGS) vektor wat noodsaaklike gene van die insek vektore van sitrusvergroeningssiekte teiken, en om bioinformaties ‘n profiel saam te stel van die virus-geïnduseerde kort inmengende RNA’s (vsiRNAs) wat in planta vervaardig word. Hierdie beheerstrategie is uitsonderlik spesifiek as gevolg van die volgordespesifieke meganisme van RNA inmenging (RNAi) masjienerie, asook die feit dat CTV ‘n floeëmbeperkte virus is. Vier potensiële gene, arginine kinase, abnormal wing disc, chitin synthase en ran-GTPase, is gekies om as teikens vir RNAi te dien. ‘n Versameling van ses insek- en geenspesifieke onderdrukkingsinvoegsels is geskep. ‘n Databasis van 24 intermediêre plasmiede, wat elk die 3’ eindpunt van CTVfl6 met een van die onderdrukkings invoegsels in die sin of teensin oriëntasie, met of sonder ‘n stroomop kopie van die CTV mantelproteïen beheerkomponent bevat, is gevestig. Die doel hiervan was om die invloed van die samestelling van die CTV VIGS vektor op vsiRNA vervaardiging in planta te evalueer. Daar is gepoog om die volledige CTVfl6 VIGS vektore aan mekaar te sit deur die 3` eindpunt van die oorspronklike genoomvolgorde te vervang met die nuutgestigte 3’ eindpunte. Slegs drie van die 24 is suksesvol gekonstrueer: twee T. erytreae insek- spesifieke, en een abnormal wing disc geen-spesifieke vektor. Al die CTVfl6 VIGS vektore het sistemiese infeksie bereik in 22% tot 33% van Nicotiana benthamiana plante 5-6 weke ná Agrobacterium tumefaciens infiltrasie. Daar is ook gepoog om Meksikaanse lemmetjie plante met twee van die drie CTVfl6 VIGS vektore te inokuleer. Kort RNA (sRNA) volgordebepaling is uitgevoer op CTV positiewe N. benthamiana materiaal, aangesien CTV infeksie nie in die meksikaanse lemmetjie plante bevestig kon word ten tyde monsters geneem is nie. Bioinformatiese analise van die N. benthamiana sRNA biblioteke het getoon dat vsiRNAs wat >99% van die CTVfl5 VIGS vektor genome dek, vervaardig is. Interaksie tussen die gasheerplant en die virus is aangedui deur ‘n toename in vsiRNA belyningsdiepte wat by twee van die geenonderdrukkers van CTV gesien kon word. Die mees betekenisvolle bevinding wat gemaak kon word deur na die samestelling van die vsiRNA profiele van die CTVfl6 VIGS vektore te kyk was dat CTVfl6 groot eksogene invoegings in die 3’ eindpunt kan hanteer. Infeksie met die CTVfl6 VIGS vektore het gelei tot die produksie van insek-spesifieke vsiRNAs in planta wat >99.78% van die onderdrukkingsinvoegsel dek. Hierdie studie was die eerste in sy soort om van hierdie RNAi strategie gebruik te maak om potensieel die endemiese Suid-Afrikaanse sitrusvergroeningssieke insek vektor, T. erytreae, te beheer. Ongeag of CLas Suid-Afrika bereik, sal die CTV VIGS vektore wat in hierdie studie geskep is toegepas kan word om die verspreiding van CLaf en sitrusvergroeningssiekte in ‘n Afrika omgewing te beperk. Masters 2022-02-07T06:28:29Z 2022-04-29T09:20:49Z 2022-02-07T06:28:29Z 2022-04-29T09:20:49Z 2022-04 Thesis http://hdl.handle.net/10019.1/124583 en_ZA Stellenbosch University xv, 89 pages : color illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Genetic vectors Biotechnology Citrus tristeza disease -- South Africa Citrus greening disease -- Control Citrus tristeza virus (CTV) -- Genetic aspects Citrus -- Diseases and pests -- Genetic aspects Small interfering RNA Gene silencing UCTD De Nicola, Ella Hannan Maria Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title | Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title_full | Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title_fullStr | Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title_full_unstemmed | Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title_short | Construction and evaluation of citrus tristeza virus-based gene silencing vectors that target Trioza erytreae and Diaphorina citri |
| title_sort | construction and evaluation of citrus tristeza virus based gene silencing vectors that target trioza erytreae and diaphorina citri |
| topic | Genetic vectors Biotechnology Citrus tristeza disease -- South Africa Citrus greening disease -- Control Citrus tristeza virus (CTV) -- Genetic aspects Citrus -- Diseases and pests -- Genetic aspects Small interfering RNA Gene silencing UCTD |
| url | http://hdl.handle.net/10019.1/124583 |
| work_keys_str_mv | AT denicolaellahannanmaria constructionandevaluationofcitrustristezavirusbasedgenesilencingvectorsthattargettriozaerytreaeanddiaphorinacitri |