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

Abstract

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 Ascochyta lentis 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 A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens 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 AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 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 AlKewell scd1::hyg 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 AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis 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.

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil

“…Gene deletion strains of P94‐24 and Al Kewell with disrupted AlAvr1‐1 and AlAvr1‐2 genes, respectively, were produced using A. tumefaciens ‐mediated transformation (ATMT) with gene‐disruption constructs pTAR‐Hyg‐ AlAvr1‐1 or pTAR‐Hyg‐ AlAvr1‐2 (Figure S10 ) as described by Debler and Henares ( 2020 ). Deletion mutants were designated Δ AlAvr1‐1 and Δ AlAvr1‐2 .…”

“…AlKewell and P94-24 were grown in yeast extract dextrose liquid medium for 3 days with shaking at 180 rpm at 22℃ and DNA isolation. Nanopore sequencing and assembly were performed as described by Debler and Henares (2020). Whole-genome alignment for comparison of A. lentis genome assemblies was performed using Nucmer v. 4.0.0 (Kurtz et al, 2004) with the "--mum" argument.…”

“…A. lentis is from the family Didymellaceae, within the Pleosporales, and provides an additional plant disease model to research molecular mechanisms of pathogen-host interaction that contrasts with the pathogens from cereal and oilseed crops described above. A near-complete genome assembly for A. lentis has been recently published (Lee et al, 2021), and agroinfiltration, genetic transformation, and gene knockout protocols are available for research in the lentil-ascochyta pathosystem (Debler & Henares, 2020;Debler et al, 2021;Henares et al, 2019). However, so far very limited information is available about the pathogen-host interaction at the molecular level.…”

“…A near‐complete genome assembly for A . lentis has been recently published (Lee et al, 2021 ), and agroinfiltration, genetic transformation, and gene knockout protocols are available for research in the lentil–ascochyta pathosystem (Debler & Henares, 2020 ; Debler et al, 2021 ; Henares et al, 2019 ). However, so far very limited information is available about the pathogen–host interaction at the molecular level.…”

“…empty vector pTAR-0-hyg(Debler & Henares, 2020) was digested with KpnI-HF at 37°C for 60 min to generate vector backbone and hygromycin selectable marker cassette fragments. Vector, selectable marker, and A. lentis target gene flanking fragments were assembled using NEBuilder HiFi DNA assembly master mix (NEB).…”