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Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi
Thesis (MScAgric)--Stellenbosch University, 2021.
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867614095394471936 |
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| access_status_str | Open Access |
| author | Lourens, Darrian Brett |
| author2 | Hills, Paul N.
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| author_browse | Hills, Paul N.
Lourens, Darrian Brett |
| author_facet | Hills, Paul N.
Lourens, Darrian Brett |
| author_sort | Lourens, Darrian Brett |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/123940 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:35.680Z |
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| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/123940 Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi Lourens, Darrian Brett Hills, Paul N. Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology. Medicago truncatula Arabinogalactan protein MtAMA1 Arbuscular mycorrhizal fungi Genotype-environment interaction UCTD Thesis (MScAgric)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Arbuscular mycorrhizal fungi (AMF) play a vital role in the health and well-being of over 90% of vascular plants. Proteins that control the novel interfaces that form between the cell walls of both the fungi and root during symbiosis are poorly understood. A group of proteins known as the arabinogalactan proteins (AGPs) have been recently shown to play a role in microbial-plant symbioses. Using the model organism Medicago truncatula, the expression of a specific AGP, MtAMA1 (Medtr5g035640), that was shown in literature to be highly expressed in only AMF- colonised roots, was tested. A low phosphate (LPM) environment, inoculated with AMF, was successful in significantly increasing the amount of mycorrhization of the roots as well as significantly increasing the abundance of arbuscules. This was in contrast to plants grown in a high phosphate (HP) environment and a high phosphate environment inoculated with AMF spores (HPM). A HP environment was able to significantly limit mycorrhization and arbuscule development even when the medium was inoculated with AMF spores. An RT-sqPCR analysis of the 18S rRNA of Glomeromycota was performed for all treatments and showed that one replicate within the LPM group had high levels of AMF-colonisation, based on DNA band intensity. The expression of MtAMA1 was tested using RT-qPCR on leaf, shoot and root material from different treatments. For the first reported time, expression of MtAMA1 was observed in all tissues, for all treatments, with variable expression between biological replicates. The variation in MtAMA1 expression, within the stem and leaf tissue did, however, follow a pattern. When a large up- regulation of MtAMA1 was observed in either the stem or leaf tissue, the other tissue (stem or leaf) had lower expression when compared between biological replicates for that specific treatment. The largest difference in MtAMA1 expression was observed for the HPM treatment. These plants appeared larger than the plants grown within the HP or LPM treatments. It is possible that the expression of MtAMA1 in aerial plant tissues could be correlated to the growth rate of a specific tissue. This is, however, speculative and future studies should test growth rate versus MtAMA1 expression. Low MtAMA1 expression was observed within the root tissue of all treatments, except for one biological replicate within the LPM treatment. This replicate had an increase in MtAMA1 expression of ~3600% when compared to the control and which correlated to the high AMF- colonisation levels observed for this specific replicate. A timepoint experiment, where plants were grown under a HP environment prior to the addition of an AMF-spore soil drench, showed that over time (0-18 d) both AMF-colonisation levels (18S rRNA DNA band intensity) and MtAMA1 gene expression increased in a linear fashion with a non-significant large positive relationship. This observation supports previous evidence from the literature and shows that high AMF colonisation levels are correlated with an upregulation of MtAMA1 in the roots. Using an eGFP- expressing vector (pCAMBIA1302-MtAMA1), MtAMA1 was shown to localize to the cell wall of Allium cepa (common onion). This observation and previous observations indicate that MtAMA1 could possibly cyto-locate to the cell wall and act as a signaling or recognition molecule. To elucidate the function of MtAMA1 further, a knockdown (pHELLSGATE12-MtAMA1-MtAMA1) and an overexpressor (pCAMBIA2300-pBinAR-MtAMA1) vector were constructed and transformed into M. truncatula via organogenesis. Due to a non-optimized kanamycin concentration in the transformation protocol, a large number of escapes were produced and no transformed overexpressor lines were generated. The process was repeated for the knockdown vector using three times the kanamycin concentration but no plants survived selection. This could indicate that a downregulation in MtAMA1 expression might induce a lethal phenotype or that the kanamycin concentration was too high even for transformants. A more economical and time saving in planta transformation protocol was tested and optimized with an empty GUS-expressing vector, pCAMBIA2301. A transformation efficiency of 30.56% was achieved, which was an improvement over the original protocol of 21.16% for the average transformation rate over several transformations and 2.66% over the highest reported transformation rate. A kanamycin kill curve was generated to provide the optimal kanamycin concentration needed for selection of transformed M. truncatula seedlings. The concentration of kanamycin needed to kill 100% of the seedlings was 357 mg/L (600 μM) for the genotype A17. The optimised in planta transformation protocol and kanamycin concentration can be used in future studies to rapidly generate M. truncatula A17 transformants. AFRKAANSE OPSOMMING: Arbuskulêre mikorrisale swamme (AMS) vertolk ʼn lewensbelangrike rol in die gesondheid en welstand van meer as 90% van alle vaskulêre plante. Proteïne wat die nuwe koppelvlakke wat vorm tussen die selwande van ablei die swam en wortel gedurende simbiose beheer, word tot dusvêr swak verstaan. ʼn Groep van samestellings bekend as die arabinogalaktaan proteïne (AGPs) was onlangs gewys om ʼn rol in mikrobiese-plant simbiose te vertolk. Met die gebruik van die modelorganisme Medicago truncatula was die uitdrukking van ʼn spesifieke AGP, MtAMA1 (Medtr5g035640), wat gewys was in literatuur om hoogs-uitgedruk te wees in slegs AMS- gekoloniseerde wortels was, getoets. ʼn Lae-fosfaat (LFM) omgewing geïnokuleer met AMS was suksesvol om die bedrag van mikorrisasie van die wortels aansienlik te verhoog, asook om die oorvloed van arubskule aansienlik te verhoog in vergelyking met plante wat in ʼn hoë-fosfaat omgewing (HF) en ʼn hoë-fosfaat omgewing geïnokuleer met AMS spore (HFM) groei. ʼn HF omgewing kon die hoeveelheid mikorrisasie en arbuskiel ontwikkeling aansienlik limiet, selfs wanneer die medium met AMS-spore geïnokuleer was. ʼn RT-sqPCR analiese van die 18S rRNA van Glomeromycota was uitgevoer vir alle behandelings en het gewys dat een herhaling binne die LFM-groep hoë vlakke van AMS-kolonisasie uitvoer, gebaseer op DNS band intensiteit. Die uitdrukking van MtAMA1 was getoets met die gebruik van RT-sqPCR op blaar, stam en wortel materiaal van verskillende behandelinge. Vir die eerste keer kan ons rapporteer dat MtAMA1 uitdrukking waargeneem in alle weevsels, vir alle behandelinge, met veranderlike uitdrukking tussen biologiese herhalings. Die variasie in MtAMA1 uitdrukking in die stam en blaar weevsel het alhoewel ʼn patroon gevolg. Wanneer hoë op-regulasie van MtAMA1 waargeneem was in óf die stam óf blaar weevsel, het die ander weevsel laer vlakke van uitdrukking gehad in vergelyking tussen biologiese herharlings vir daardie spesifieke behandeling. Die grootste verskil in MtAMA1 uitdrukking was waargeneem vir die HFM-behandeling. Hierdie plante was anekdoties groter as die plante in die HF of LFM behandelinge. Dit is moontlik dat die uitdrukking van MtAMA1 in bo-grondse plantweevsels gekorrileer kan wees aan die groeikoers van ʼn spesifieke weevsel. Hierdie is alhoewel spekulatief, en toekomstige studies moet groeikoers teenoor MtAMA1 uitdrukking toets. Lae MtAMA1 uitdrukking was waargeneem in die wortelweevsel van alle behandelinge, behalwe vir een biologiese herhaling binne die LFM behandeling. Hierdie replikaat het ʼn toename in MtAMA1 uitdrukking van ~3600% gehad in vergelyking met die kontrole, en wat gekorreleer het aan die hoë AMF-kolonisasie vlakke waargeneem vir hierdie spesifieke replikaat. ʼn Tydspunt eksperiment, waar plante gegroei was onder ʼn HF omgewing voor die byvoeg van ʼn AMS-spoor grond deurdrenk, het gewys dat oor tyd (0-18 d) het albei die AMS kolonisasievlakke (18S rRNA DS band intensiteit) en MtAMA1 geenuitdrukking toegeneem in ʼn lineêre mode met ʼn nie-betekenisvolle groot positiewe verhouding. Hierdie waarneming ondersteun vorige bewyse in die literatuur en wys dat hoë AMS kolonisasievlakke veroorsaak ʼn opregulasie van MtAMA1 in die wortels. Met die gebruik van ʼn eGFP-uitdrukkende vektor (pCAMBIA1302- MtAMA1) was MtAMA1 gewys om in die selwand van Allium cepa (algemene ui) te lokaliseer. Hierdie waarneeming, asook vorige waarnemings, dui aan dat MtAMA1 moontlik sito-lokaliseer na die selwand en ʼn rol as ʼn sein of herkennings molekule speel. Om die funksie van MtAMA1 vêrder toe te lig, was ʼn aflsaan (pHELLSGATE12-MtAMA1-MtAMA1) en ʼn ooruitdrukker (pCAMBIA2300-pBinAR-MtAMA1) vektor gebou en in M. truncatula getransformeer deur organogenese. As gevolg van ʼn nie-geoptimaliseerde kanamycin konsentrasie in die transformasie protokol, was ʼn groot bedrag van ontsnappings geproduseer en geen getransformeerde ooruitdrukker lyne was gegenereer nie. Hierdie proses was herhaal vir die afslaan vektor met drie keer die kanamycin konsentrasie, maar geen ontsnappings was waargeneem nie en geen plante het seleksie oorleef nie. Hierdie kan aandui dat ʼn afregulasie in MtAMA1 uitdrukking dalk ʼn dodelike fenotiepe kan veroorsaak, of dat die kanamycin konsentrasie te sterk was selfs vir die getransformeerde weevsels. ʼn Meer ekonomiese en tydsbesparende in planta transformasie protokol was getoets en geoptimaliseer met ʼn leë GUS-uitdrukkende vektor pCAMBIA2301. ʼn Transformasie doeltreffenheid van 30.56% was bereik, wat ʼn verbetering oor die oorspronklike protokol van 21.16% vir die gemiddelde transformasiekoers en 2.66% oor die hoogste gerapporteerde transformasiekoers. ʼn Kanamycin doodmaak-kurwe was gegenereer om die optimale kanamycing konsentrasie benodig vir seleksie van getransformeerde M. truncatula saailinge te verskaf. Die konsentrasie van kanamycin benodig om 100% van saailinge dood te maak was 357 mg/L (600 μM) vir die genotipe A17. Die geoptimaliseerde in planta transformasie protokol en kanamycin konsentrasie kan in toekomstige studies gebruik word om vinnig M. truncatula A17 transformante te genereer. Masters 2021-12-06T09:51:33Z 2021-12-22T14:30:22Z 2021-12-06T09:51:33Z 2021-12-22T14:30:22Z 2021-12 Thesis http://hdl.handle.net/10019.1/123940 en_ZA Stellenbosch University xvi, 101 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Medicago truncatula Arabinogalactan protein MtAMA1 Arbuscular mycorrhizal fungi Genotype-environment interaction UCTD Lourens, Darrian Brett Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title | Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title_full | Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title_fullStr | Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title_full_unstemmed | Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title_short | Determining the function of MtAMA1, an AGP suspected to play a role in the symbiosis between Medicago truncatula and arbuscular mycorrhizal fungi |
| title_sort | determining the function of mtama1 an agp suspected to play a role in the symbiosis between medicago truncatula and arbuscular mycorrhizal fungi |
| topic | Medicago truncatula Arabinogalactan protein MtAMA1 Arbuscular mycorrhizal fungi Genotype-environment interaction UCTD |
| url | http://hdl.handle.net/10019.1/123940 |
| work_keys_str_mv | AT lourensdarrianbrett determiningthefunctionofmtama1anagpsuspectedtoplayaroleinthesymbiosisbetweenmedicagotruncatulaandarbuscularmycorrhizalfungi |