Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>

ABSTRACT The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantita...

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Autores principales:	Jingtao Li, Yanhua Zhang, Yucheng Zhang, Pei-Ling Yu, Hongyu Pan, Jeffrey A. Rollins
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR Cas9 filamentous fungi functional genomics gene disruption necrotroph Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ecd9af93cc17434eaa07d5dccfbf0716
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spelling	oai:doaj.org-article:ecd9af93cc17434eaa07d5dccfbf07162021-11-15T16:00:26ZIntroduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>10.1128/mBio.00567-182150-7511https://doaj.org/article/ecd9af93cc17434eaa07d5dccfbf07162018-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00567-18https://doaj.org/toc/2150-7511ABSTRACT The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function. IMPORTANCE Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. Sclerotinia sclerotiorum is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in S. sclerotiorum, we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.Jingtao LiYanhua ZhangYucheng ZhangPei-Ling YuHongyu PanJeffrey A. RollinsAmerican Society for MicrobiologyarticleCRISPRCas9filamentous fungifunctional genomicsgene disruptionnecrotrophMicrobiologyQR1-502ENmBio, Vol 9, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Cas9 filamentous fungi functional genomics gene disruption necrotroph Microbiology QR1-502
spellingShingle	CRISPR Cas9 filamentous fungi functional genomics gene disruption necrotroph Microbiology QR1-502 Jingtao Li Yanhua Zhang Yucheng Zhang Pei-Ling Yu Hongyu Pan Jeffrey A. Rollins Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
description	ABSTRACT The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function. IMPORTANCE Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. Sclerotinia sclerotiorum is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in S. sclerotiorum, we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.
format	article
author	Jingtao Li Yanhua Zhang Yucheng Zhang Pei-Ling Yu Hongyu Pan Jeffrey A. Rollins
author_facet	Jingtao Li Yanhua Zhang Yucheng Zhang Pei-Ling Yu Hongyu Pan Jeffrey A. Rollins
author_sort	Jingtao Li
title	Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
title_short	Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
title_full	Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
title_fullStr	Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
title_full_unstemmed	Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>
title_sort	introduction of large sequence inserts by crispr-cas9 to create pathogenicity mutants in the multinucleate filamentous pathogen <named-content content-type="genus-species">sclerotinia sclerotiorum</named-content>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/ecd9af93cc17434eaa07d5dccfbf0716
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Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen <named-content content-type="genus-species">Sclerotinia sclerotiorum</named-content>

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