New capabilities for Mycosphaerella graminicola research

Abstract

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.

Transcriptome and Metabolite Profiling of the Infection Cycle ofZymoseptoria triticion Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifestyle Definition

“…Agrobacterium tumefaciens-mediated fungal transformation was performed as described previously (Motteram et al, 2009) using the Ku70 modified form of isolate IPO323 (Bowler et al, 2010). Constructs designed to either delete or disrupt candidate secreted protein effector genes were generated in vector pCHYG (Motteram et al, 2009).…”

The Genomes of Mycosphaerella graminicola and M. fijiensis

“…For M. graminicola, this problem has been overcome for the most part by knocking out the ku70 gene (Bowler et al 2010); frequencies of homologous recombination of transformants into a ku70 disruption strain can be as high as 95 % with no obvious effects on wild-type growth. This makes high-throughput transformation for functional analysis of genes in this species feasible.…”

Analysis of Two in Planta Expressed LysM Effector Homologs from the FungusMycosphaerella graminicolaReveals Novel Functional Properties and Varying Contributions to Virulence on Wheat

“…M. graminicola transformation was performed in a DKu70-modified strain that typically supports homologous recombination frequencies of greater than 70% (Bowler et al, 2010). Three and four independent strains for Mg1LysM and Mg3LysM, respectively, were confirmed to have single T-DNA integrations within the target locus via Southern analysis (Supplemental Fig.…”

“…This modified strain carries a deletion of the Ku70 gene, which otherwise facilitates nonhomologous recombination events and increases the rate of generating homologous recombinants via transformation to greater than 70% (Bowler et al, 2010). All strains were stored at 280°C in 50% (v/v) glycerol.…”