Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event
Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as report...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2019-12-01
|
| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/8/1/54 |
| _version_ | 1818267405118341120 |
|---|---|
| author | Inoka K. Hettiarachchige Emma J. Ludlow Piyumi N. Ekanayake Natasha D. Brohier Sareena Sahab Timothy I. Sawbridge German C. Spangenberg Kathryn M. Guthridge |
| author_facet | Inoka K. Hettiarachchige Emma J. Ludlow Piyumi N. Ekanayake Natasha D. Brohier Sareena Sahab Timothy I. Sawbridge German C. Spangenberg Kathryn M. Guthridge |
| author_sort | Inoka K. Hettiarachchige |
| collection | DOAJ |
| description | Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (<i>Lolium perenne</i> L.)-associated <i>Epichloë</i> endophyte strains using <i>A. tumefaciens</i>-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail. |
| first_indexed | 2024-12-12T20:22:04Z |
| format | Article |
| id | doaj.art-d9c6b42d987f4b0a97996b70eeeca6cf |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-12-12T20:22:04Z |
| publishDate | 2019-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-d9c6b42d987f4b0a97996b70eeeca6cf2022-12-22T00:13:14ZengMDPI AGMicroorganisms2076-26072019-12-01815410.3390/microorganisms8010054microorganisms8010054Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration EventInoka K. Hettiarachchige0Emma J. Ludlow1Piyumi N. Ekanayake2Natasha D. Brohier3Sareena Sahab4Timothy I. Sawbridge5German C. Spangenberg6Kathryn M. Guthridge7Agriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaMethods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (<i>Lolium perenne</i> L.)-associated <i>Epichloë</i> endophyte strains using <i>A. tumefaciens</i>-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.https://www.mdpi.com/2076-2607/8/1/54<i>epichloë</i>endophytereporter genegreen fluorescent proteindsred<i>a. tumefaciens</i>-mediated transformationtransformantst-dna integrationsequencing |
| spellingShingle | Inoka K. Hettiarachchige Emma J. Ludlow Piyumi N. Ekanayake Natasha D. Brohier Sareena Sahab Timothy I. Sawbridge German C. Spangenberg Kathryn M. Guthridge Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event Microorganisms <i>epichloë</i> endophyte reporter gene green fluorescent protein dsred <i>a. tumefaciens</i>-mediated transformation transformants t-dna integration sequencing |
| title | Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event |
| title_full | Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event |
| title_fullStr | Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event |
| title_full_unstemmed | Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event |
| title_short | Generation of <i>Epichloë</i> Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event |
| title_sort | generation of i epichloe i strains expressing fluorescent proteins suitable for studying host endophyte interactions and characterisation of a t dna integration event |
| topic | <i>epichloë</i> endophyte reporter gene green fluorescent protein dsred <i>a. tumefaciens</i>-mediated transformation transformants t-dna integration sequencing |
| url | https://www.mdpi.com/2076-2607/8/1/54 |
| work_keys_str_mv | AT inokakhettiarachchige generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT emmajludlow generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT piyuminekanayake generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT natashadbrohier generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT sareenasahab generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT timothyisawbridge generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT germancspangenberg generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent AT kathrynmguthridge generationofiepichloeistrainsexpressingfluorescentproteinssuitableforstudyinghostendophyteinteractionsandcharacterisationofatdnaintegrationevent |