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Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions
Published on: September 30, 2016
An Efficient Strategy for Obtaining Mutants by Targeted Gene Deletion in Ophiostoma novo-ulmi
Jorge Luis Sarmiento-Villamil1, Thais Campos de Oliveira1, Erika Sayuri Naruzawa1,2
1Centre d'Étude de la Forêt (CEF) and Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Quebec City, QC, Canada.
Abstract:
The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current, highly destructive, pandemic of Dutch elm disease (DED). Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes, which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens. Then, O. novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the "model species" Candida albicans and Ustilago maydis. In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease, we developed a high-throughput gene deletion protocol for O. novo-ulmi. The protocol is based on transforming a Δmus52 O. novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain, and transforming this strain with the latest version of OSCAR plasmids. The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA. The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT). To validate the high efficiency of our deletion gene methodology we deleted ade7, which also belongs to the purine nucleotide pathway, as well as bct2, ogf1, and opf2 which encode fungal binuclear transcription factors (TFs). The frequency of gene replacement by HR for these genes reached up to 94%. We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O. novo-ulmi genes and other filamentous fungi.
Insights
A new high-throughput gene deletion protocol for Ophiostoma novo-ulmi, the fungus causing Dutch elm disease, was developed. This method significantly improves gene deletion efficiency for studying fungal pathogens.
Area of Science:
- Mycology
- Plant Pathology
- Molecular Biology
Background:
- Ophiostoma novo-ulmi is a dimorphic fungus causing destructive Dutch elm disease (DED).
- Its pathogenic mechanisms and dimorphic transition involve unique molecular pathways distinct from model species like Candida albicans.
- Understanding these pathways is crucial for developing disease management strategies.
Purpose of the Study:
- To develop a high-throughput gene deletion protocol for Ophiostoma novo-ulmi.
- To facilitate the study of molecular mechanisms underlying vascular wilt diseases caused by dimorphic fungi.
- To enable efficient genetic manipulation of O. novo-ulmi for research.
Main Methods:
- Utilized a non-homologous end joining (NHEJ) deficient mutant (Δmus52) of O. novo-ulmi as the recipient strain.
- Employed OSCAR plasmids containing the Herpes simplex virus thymidine kinase (HSVtk) gene to prevent ectopic integration.
- Validated the protocol by targeting genes involved in purine nucleotide biosynthesis (ade1, ade7) and transcription factors (bct2, ogf1, opf2).
Main Results:
- Achieved high gene deletion frequencies via homologous recombination (HR): up to 77.8% for ade1 in the Δmus52 mutant, compared to 2% in wild-type.
- Demonstrated high efficiency for other gene targets, with frequencies reaching up to 94% for ade7, bct2, ogf1, and opf2.
- The developed protocol significantly enhances the efficiency of gene deletion in O. novo-ulmi.
Conclusions:
- The novel methodology combining NHEJ-deficient strains and OSCAR plasmids is highly efficient for gene deletion in O. novo-ulmi.
- This protocol is expected to be applicable to other genes within O. novo-ulmi and across various filamentous fungi.
- Provides a powerful tool for advancing research into the molecular biology of fungal pathogens and vascular wilt diseases.
