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

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.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System

“…These expression cassettes were delivered by various transformation methods in fungi and oomycetes. For filamentous fungi, there are four commonly used methods: (i) exogenous DNA introduction using PEG solution into protoplasts generated by cell wall lysing enzymes (protoplast mediated transformation, PMT) ( Nødvig et al, 2015 ), (ii) Agrobacterium tumefaciens co-cultivation for transferring vector DNA (AMT) ( Zou et al, 2020 ) ( Figure 1 ), (iii) reversible membrane permeabilization induced by local application of electric pulses (Electroporation, EP), and (iv) tungsten particles coated with DNA, accelerated to a high velocity, and introduced into cells (Biolistic transformation, BT) ( Wang, 2018 ). In oomycetes, protoplast- or AMT methods have been mainly used for delivering the CRISPR/Cas9-guide RNA constructs into the cells ( Fang and Tyler, 2016 ; Gumtow et al, 2018 ).…”

“…Mutants of plant pathogens with variable pathogenicity from wild type can be generated using the CRISPR/Cas9 system. Zou et al (2020) described a point mutation in the avirulence gene of Leptosphaeria maculans 7 ( AvrLm7 ) that resulted in a shift from an avirulent strain ( UMAvr7 ) to a virulent strain ( umavr7 ) on the Brassica napus Rlm7 genotype. This pathogen L. maculans causes a severe disease called blackleg in canola ( B. napus ).…”

Gene editing in Brassica napus for basic research and trait development

“…In a disease survey conducted in western Canada, a total of 180 Avr gene races were identified from 964 isolates, with three major races observed: AvrLm-2-45-6-7, AvrLm2-4-5-6-7-S, and AvrLm-1-4-5-6-7-11-(S). The UMAvr7 isolate carried AvrLm1-2-3-4-9-11-LepR1LepR2-S-AvrLm5-6-7 and is PCR positive for AvrLm5, AvrLm6, and AvrLm4-7 (Zou et al 2020). Previously, Parlange et al (2009) determined that when codon358 in AvrLm4-7 has a transversion of "C" to "G", this pathogen is recognized by Rlm7 and Rlm4/7 in canola.…”

“…Previously, Parlange et al (2009) determined that when codon358 in AvrLm4-7 has a transversion of "C" to "G", this pathogen is recognized by Rlm7 and Rlm4/7 in canola. Zou et al (2020) used Agrobacterium transformation of L. maculans isolate (DS103) (named UMAvr7) to make LOF alleles of the avirulence gene target AvrLm4-7 as previously described by Gardiner and Howlett (2004). The resulting mutant isolates were tested on different canola genotypes with 6 plants per genotype inoculated as described by Zhang et al (2016).…”

“…Westar, indicating a shift from avirulence to virulence. Further, by assessing H 2 O 2 accumulation in cotyledons of resistant genotype 01-23-2-1, UMAvr7 caused limited accumulation around the wound site compared to mutant isolate umavr7 (Zou et al 2020).…”