Cargando…
Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi
The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock perfor...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922272/ https://www.ncbi.nlm.nih.gov/pubmed/33669319 http://dx.doi.org/10.3390/toxins13020153 |
_version_ | 1783658650588413952 |
---|---|
author | Florea, Simona Jaromczyk, Jolanta Schardl, Christopher L. |
author_facet | Florea, Simona Jaromczyk, Jolanta Schardl, Christopher L. |
author_sort | Florea, Simona |
collection | PubMed |
description | The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala—a triploid hybrid species—were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement. |
format | Online Article Text |
id | pubmed-7922272 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-79222722021-03-03 Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi Florea, Simona Jaromczyk, Jolanta Schardl, Christopher L. Toxins (Basel) Article The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala—a triploid hybrid species—were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement. MDPI 2021-02-16 /pmc/articles/PMC7922272/ /pubmed/33669319 http://dx.doi.org/10.3390/toxins13020153 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Florea, Simona Jaromczyk, Jolanta Schardl, Christopher L. Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title | Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title_full | Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title_fullStr | Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title_full_unstemmed | Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title_short | Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi |
title_sort | non-transgenic crispr-mediated knockout of entire ergot alkaloid gene clusters in slow-growing asexual polyploid fungi |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922272/ https://www.ncbi.nlm.nih.gov/pubmed/33669319 http://dx.doi.org/10.3390/toxins13020153 |
work_keys_str_mv | AT floreasimona nontransgeniccrisprmediatedknockoutofentireergotalkaloidgeneclustersinslowgrowingasexualpolyploidfungi AT jaromczykjolanta nontransgeniccrisprmediatedknockoutofentireergotalkaloidgeneclustersinslowgrowingasexualpolyploidfungi AT schardlchristopherl nontransgeniccrisprmediatedknockoutofentireergotalkaloidgeneclustersinslowgrowingasexualpolyploidfungi |