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Updated: Dec 10, 2025
Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira
Published on: August 14, 2021
Generation and Characterization of a Virulent Leptosphaeria maculans Isolate Carrying a Mutated AvrLm7 Gene Using the
Zhongwei Zou1, Fei Liu1, Carrie Selin1
1Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada.
Abstract:
Blackleg, caused by the fungal pathogen Leptosphaeria 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 the AvrLm7 coding region in a L. maculans isolate (UMAvr7). Reverse transcription PCR and transcriptome data confirmed that the AvrLm7 gene was knocked out in the mutant isolate. Pathogenicity tests indicated that umavr7 can cause large lesions on a set of Brassica differential genotypes that express different resistance (R) genes. Comparative pathogenicity tests between UMAvr7 (wild type) and umavr7 on the corresponding B. napus genotype 01-23-2-1 (with Rlm7) 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 B. napus Rlm7 genotype. Therefore, this mutant is virulence on the identified resistant genes to blackleg disease in B. napus genotypes. Superoxide accumulated differently in cotyledons in response to infection with UMAvr7 and umavr7, especially in resistant B. napus genotype 01-23-2-1. Resistance/susceptibility was further evaluated on 123 B. napus genotypes with the mutant isolate, umavr7. Only 6 of the 123 genotypes showed resistance to umavr7. The identification of these six resistant B. napus genotypes will lead to further studies on the development of blackleg disease resistance through breeding and the identification of novel R genes.
Insights
Researchers used CRISPR gene editing to create a mutant fungal strain that overcomes canola blackleg resistance. This discovery aids in developing new disease-resistant crops and identifying novel resistance genes.
Area of Science:
- Plant Pathology
- Molecular Genetics
- Crop Science
Background:
- Blackleg, caused by *Leptosphaeria maculans*, is a major threat to global canola (*Brassica napus*) production.
- Understanding pathogen virulence factors and plant resistance mechanisms is crucial for disease management.
Purpose of the Study:
- To generate and characterize a mutant *L. maculans* isolate with altered virulence using CRISPR/Cas9 technology.
- To investigate the shift in pathogenicity and identify new sources of resistance in *B. napus*.
Main Methods:
- CRISPR/Cas9 gene editing was used to knock out the *AvrLm7* gene in *L. maculans*.
- Reverse transcription PCR and transcriptome analysis confirmed gene knockout.
- Pathogenicity tests were conducted on differential *Brassica* genotypes and 123 *B. napus* lines.
Main Results:
- A mutant isolate, umavr7, with a knocked-out *AvrLm7* gene was successfully generated.
- umavr7 exhibited a shift from avirulent to virulent pathogenicity on *B. napus* genotypes carrying the *Rlm7* resistance gene.
- Differential superoxide accumulation was observed in response to wild-type and mutant infections.
- Only 6 out of 123 tested *B. napus* genotypes showed resistance to umavr7.
Conclusions:
- The generated mutant isolate provides a valuable tool for studying blackleg-plant interactions.
- Identification of resistant *B. napus* genotypes opens avenues for breeding programs and novel resistance gene discovery.
- This research contributes to developing sustainable strategies for managing blackleg disease in canola.
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