Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete <i>Ascochyta lentis</i> which causes b...

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Main Authors: Johannes W. Debler, Bernadette M. Henares
Format: Article
Language:English
Published: MDPI AG 2020-11-01
Series:Journal of Fungi
Subjects:
Online Access:https://www.mdpi.com/2309-608X/6/4/314
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spelling doaj-228bb319352043b4bc27b083135e587d2020-11-27T08:08:31ZengMDPI AGJournal of Fungi2309-608X2020-11-01631431410.3390/jof6040314Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>Johannes W. Debler0Bernadette M. Henares1Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA 6102, AustraliaCentre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA 6102, AustraliaSustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete <i>Ascochyta lentis</i> which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of <i>A. lentis</i>, we developed a targeted gene replacement method using <i>Agrobacterium tumefaciens</i> mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in <i>Al</i>Kewell. Two <i>SCD</i> genes have been identified in <i>Al</i>Kewell, <i>AlSCD1</i> and <i>AlSCD2</i>. Phylogenetic analysis revealed that <i>AlSCD1</i> clustered with the previously characterized fungal SCDs; thus, <i>AlSCD1</i> was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting <i>Al</i>Kewell <i>scd1::hyg</i> transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of <i>AlSCD1</i> gene did not result in a change in the virulence profile of <i>Al</i>Kewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in <i>A. lentis</i> which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.https://www.mdpi.com/2309-608X/6/4/314<i>Ascochyta lentis</i>scytalone dehydratasegenetic manipulationDHN-melanin biosynthesis<i>Agrobacterium tumefaciens</i>-mediated transformation
collection DOAJ
language English
format Article
sources DOAJ
author Johannes W. Debler
Bernadette M. Henares
spellingShingle Johannes W. Debler
Bernadette M. Henares
Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
Journal of Fungi
<i>Ascochyta lentis</i>
scytalone dehydratase
genetic manipulation
DHN-melanin biosynthesis
<i>Agrobacterium tumefaciens</i>-mediated transformation
author_facet Johannes W. Debler
Bernadette M. Henares
author_sort Johannes W. Debler
title Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
title_short Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
title_full Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
title_fullStr Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
title_full_unstemmed Targeted Disruption of Scytalone Dehydratase Gene Using <i>Agrobacterium tumefaciens</i>-Mediated Transformation Leads to Altered Melanin Production in <i>Ascochyta lentis</i>
title_sort targeted disruption of scytalone dehydratase gene using <i>agrobacterium tumefaciens</i>-mediated transformation leads to altered melanin production in <i>ascochyta lentis</i>
publisher MDPI AG
series Journal of Fungi
issn 2309-608X
publishDate 2020-11-01
description Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete <i>Ascochyta lentis</i> which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of <i>A. lentis</i>, we developed a targeted gene replacement method using <i>Agrobacterium tumefaciens</i> mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in <i>Al</i>Kewell. Two <i>SCD</i> genes have been identified in <i>Al</i>Kewell, <i>AlSCD1</i> and <i>AlSCD2</i>. Phylogenetic analysis revealed that <i>AlSCD1</i> clustered with the previously characterized fungal SCDs; thus, <i>AlSCD1</i> was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting <i>Al</i>Kewell <i>scd1::hyg</i> transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of <i>AlSCD1</i> gene did not result in a change in the virulence profile of <i>Al</i>Kewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in <i>A. lentis</i> which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.
topic <i>Ascochyta lentis</i>
scytalone dehydratase
genetic manipulation
DHN-melanin biosynthesis
<i>Agrobacterium tumefaciens</i>-mediated transformation
url https://www.mdpi.com/2309-608X/6/4/314
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AT bernadettemhenares targeteddisruptionofscytalonedehydratasegeneusingiagrobacteriumtumefaciensimediatedtransformationleadstoalteredmelaninproductioniniascochytalentisi
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