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

Blackleg, caused by the fungal pathogenLeptosphaeria maculans, is the most important disease affecting canola (Brassica napus) 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 theAvrLm7coding region in aL. maculansisolate (UMAvr7). Reverse transcription PCR and transcriptome data confirmed that theAvrLm7gene was knocked out in the mutant isolate. Pathogenicity tests indicated that umavr7 can cause large lesions on a set ofBrassicadifferential genotypes that express different resistance (R) genes. Comparative pathogenicity tests between UMAvr7 (wild type) and umavr7 on the correspondingB. napusgenotype 01-23-2-1 (withRlm7) 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 theB. napus Rlm7genotype. Therefore, this mutant is virulence on the identified resistant genes to blackleg disease inB. napusgenotypes. Superoxide accumulated differently in cotyledons in response to infection with UMAvr7 and umavr7, especially in resistantB. napusgenotype 01-23-2-1. Resistance/susceptibility was further evaluated on 123B. napusgenotypes with the mutant isolate, umavr7. Only 6 of the 123 genotypes showed resistance to umavr7. The identification of these six resistantB. napusgenotypes will lead to further studies on the development of blackleg disease resistance through breeding and the identification of novelRgenes.