AUTHOR=Zou Zhongwei , Liu Fei , Selin Carrie , Fernando W. G. Dilantha 

TITLE=Generation and Characterization of a Virulent Leptosphaeria maculans Isolate Carrying a Mutated AvrLm7 Gene Using the CRISPR/Cas9 System

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01969

DOI=10.3389/fmicb.2020.01969

ISSN=1664-302X

ABSTRACT=<p>Blackleg, caused by the fungal pathogen <italic>Leptosphaeria maculans</italic>, is the most important disease affecting canola (<italic>Brassica napus</italic>) crops worldwide. We employed the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to generate the mutant isolate umavr7 from a point mutation of the <italic>AvrLm7</italic> coding region in a <italic>L. maculans</italic> isolate (UMAvr7). Reverse transcription PCR and transcriptome data confirmed that the <italic>AvrLm7</italic> gene was knocked out in the mutant isolate. Pathogenicity tests indicated that umavr7 can cause large lesions on a set of <italic>Brassica</italic> differential genotypes that express different resistance (<italic>R</italic>) genes. Comparative pathogenicity tests between UMAvr7 (wild type) and umavr7 on the corresponding <italic>B. napus</italic> genotype 01-23-2-1 (with <italic>Rlm7</italic>) showed that umavr7 is a mutant isolate, producing large gray/green lesions on cotyledons. The pathogenicity of the mutant isolate was shifted from avirulent to virulent on the <italic>B. napus Rlm7</italic> genotype. Therefore, this mutant is virulence on the identified resistant genes to blackleg disease in <italic>B. napus</italic> genotypes. Superoxide accumulated differently in cotyledons in response to infection with UMAvr7 and umavr7, especially in resistant <italic>B. napus</italic> genotype 01-23-2-1. Resistance/susceptibility was further evaluated on 123 <italic>B. napus</italic> genotypes with the mutant isolate, umavr7. Only 6 of the 123 genotypes showed resistance to umavr7. The identification of these six resistant <italic>B. napus</italic> genotypes will lead to further studies on the development of blackleg disease resistance through breeding and the identification of novel <italic>R</italic> genes.</p>