2019 Summer Undergraduate Research Program (SURP)

Presentation Type

Open Access Poster Presentation

Keywords

Fusarium; Gene editing; CRISPR-assciated protein 9;

Abstract

Fusarium graminearum is a pathogenic fungus that causes scab or Fusarium head blight disease in barley and wheat. The disease not only reduces yield, but it also contaminates the kernel with harmful toxins. Therefore, the disease causes economic loss and poses a health risk to humans and animals (McMullen et al., 2012). There is no natural resistance to scab disease in barley and there are no effective fungicides to control it. Alternative approaches to protect barley from this devastating disease are badly needed. In recent years, an RNA-guided genome-editing system known as clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 9 (CRISPR/Cas9), has become a popular method for genome modification (Jinek et al., 2012). This technology is a modification of the defense mechanism that many bacteria and archaea use to protect themselves against invading DNA viruses. CRISPR/Cas9 can be used to mutate genomes and make a pathogenic organism less infectious. During the 2019 SURP, we tested whether mutating F. graminearum with CRISPR/Cas9 makes it less infectious. We used CRISPR/Cas9 plasmids to mutate three genes in F. graminearum. One of the genes (AUR1) is a visual marker and the other two genes (MGV1 and Tri5) are essential for infection. The CRISPR/Cas9 plasmids also contained a hygromycin B resistance gene for selection. We transformed F. graminearum protoplasts and selected colonies on media containing hygromycin B. We hypothesized that transforming F. graminearum protoplasts with CRISPR/Cas9 plasmids would edit the target genes. We recovered many colonies on selective media. This suggests that colonies carry the CRISPR/Cas9 plasmids. However, we were unable to detect plasmid DNA in the colonies we screened by PCR. This might be due to the fact that F. graminearum is multinucleated and not all nuclei are transformed. Further research is needed to make the screening process more efficient in order to isolate homokaryons (cells with multiple identical nuclei).

Start Date

2-8-2019 11:30 AM

End Date

2-8-2019 1:30 PM

Event Host

Summer Undergraduate Research Program, University of Northern Iowa

Faculty Advisor

Tilahun Abebe

Department

Department of Biology

File Format

application/pdf

Included in

Genetics Commons

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Aug 2nd, 11:30 AM Aug 2nd, 1:30 PM

Editing Fusarium graminearum genome with CRISPR/Cas9

Fusarium graminearum is a pathogenic fungus that causes scab or Fusarium head blight disease in barley and wheat. The disease not only reduces yield, but it also contaminates the kernel with harmful toxins. Therefore, the disease causes economic loss and poses a health risk to humans and animals (McMullen et al., 2012). There is no natural resistance to scab disease in barley and there are no effective fungicides to control it. Alternative approaches to protect barley from this devastating disease are badly needed. In recent years, an RNA-guided genome-editing system known as clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 9 (CRISPR/Cas9), has become a popular method for genome modification (Jinek et al., 2012). This technology is a modification of the defense mechanism that many bacteria and archaea use to protect themselves against invading DNA viruses. CRISPR/Cas9 can be used to mutate genomes and make a pathogenic organism less infectious. During the 2019 SURP, we tested whether mutating F. graminearum with CRISPR/Cas9 makes it less infectious. We used CRISPR/Cas9 plasmids to mutate three genes in F. graminearum. One of the genes (AUR1) is a visual marker and the other two genes (MGV1 and Tri5) are essential for infection. The CRISPR/Cas9 plasmids also contained a hygromycin B resistance gene for selection. We transformed F. graminearum protoplasts and selected colonies on media containing hygromycin B. We hypothesized that transforming F. graminearum protoplasts with CRISPR/Cas9 plasmids would edit the target genes. We recovered many colonies on selective media. This suggests that colonies carry the CRISPR/Cas9 plasmids. However, we were unable to detect plasmid DNA in the colonies we screened by PCR. This might be due to the fact that F. graminearum is multinucleated and not all nuclei are transformed. Further research is needed to make the screening process more efficient in order to isolate homokaryons (cells with multiple identical nuclei).