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CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

journal contribution
posted on 18.01.2022, 05:24 by M Rocafort, S Arshed, D Hudson, JS Sidhu, JK Bowen, Kim PlummerKim Plummer, RE Bradshaw, RD Johnson, LJ Johnson, CH Mesarich
Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Funding

MR and CHM are supported by the Marsden Fund Council from Government funding (project ID 17-MAU-100), managed by Royal Society Te Aparangi. SA and JKB received funding from The New Zealand Institute for Plant and Food Research Limited, Strategic Science Investment Fund, Project number: 12070. DH, JS, RDJ and LJJ are supported by the MBIE partnership programme: Novel variation for a persistent problem (project ID C10X1902). AgResearch work was also supported through a QEII technicians’ study award (to JS) and the New Zealand Strategic Science Investment Fund (SSIF) contract A20067.

History

Publication Date

01/01/2022

Journal

Fungal Biology

Volume

126

Issue

1

Pagination

12p. (p.35-46)

Publisher

Elsevier

ISSN

1878-6146

Rights Statement

© 2021 British Mycological Society. Published by Elsevier Ltd. All rights reserved.