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...
Main Authors: | , |
---|---|
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 |
id |
doaj-228bb319352043b4bc27b083135e587d |
---|---|
record_format |
Article |
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 |
work_keys_str_mv |
AT johanneswdebler targeteddisruptionofscytalonedehydratasegeneusingiagrobacteriumtumefaciensimediatedtransformationleadstoalteredmelaninproductioniniascochytalentisi AT bernadettemhenares targeteddisruptionofscytalonedehydratasegeneusingiagrobacteriumtumefaciensimediatedtransformationleadstoalteredmelaninproductioniniascochytalentisi |
_version_ |
1724413673794961408 |