Date on Senior Honors Thesis

5-2021

Document Type

Senior Honors Thesis

Degree Name

B.S.

Department

Biology

Degree Program

College of Arts and Sciences

Author's Keywords

Microbotryum dianthorum; Dianthus pavonius; transformation; smut fungus; Gibson assembly

Abstract

Microbotryum dianthorum is a species of smut fungus which causes infection in plants in the Dianthus genus. The infection process is aided by small-secreted proteins known as effectors. Effectors may aid in the infection process because they are secreted by the fungus and can enter plant tissue, possibly to facilitate infection and/or manipulate the host. Thus, it is of interest to investigate the genes for these effectors to determine their role in the infection process. One method to study genes is through gene disruption, via the CRISPR/Cas9 gene editing system, but this has not yet been implemented in Microbotryum dianthorum. The goal of this study was to establish reliable CRISPR/Cas9 gene editing in Microbotryum species, so in the future it can be used to target genes of putative effectors. A vector containing cas9 was constructed and was successfully used to transform Microbotryum dianthorum. The vector was designed to allow controlled expression of cas9, such that expression should be high with rich media and much reduced with low-nutrient media. This prediction was evaluated by measuring transcription levels using Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). It did not appear, from qRT-PCR, that expression was regulated by nutrient levels as previously expected. Nevertheless, the data did confirm that the cas9 gene was expressed in at least one Microbotryum species, paving the way for implementation of CRISPR/Cas9 targeted gene disruptions in this group of fungi.

Lay Summary

Microbotryum dianthorum is a species of fungus that infects Dianthus flowers (wildflower species in the Carnation family). It is believed that the fungi can infect the flowers by using small secreted proteins. These proteins can enter the plant tissue and may help with infection or make changes in the host plant. In this project we wanted to create a reliable way to investigate the genes that encode these small secreted proteins. The way we attempted to do this was by putting the CRISPR/Cas9 gene editing system in the fungus. CRISPR/Cas9 gene editing had not yet been used in the Microbotryum genus, in particular, Microbotryum dianthorum, so we wanted to create a way to reliably be able to do this so eventually gene editing could be done on the genes for the small secreted proteins. We put a promoter that was thought to change expression levels of the genes it controls based on nutrient availability and cas9 in a plasmid. Thus, if grown on a high nutrient media, cas9 would be expressed at a higher level and if grown on a low nutrient media, cas9 would be expressed a very low level. After we successfully got the plasmid into Microbotryum we tested the expression levels on the low and high media using Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). The results from this test seemed to show that the promoter was not changing expression of cas9 based on media type but cas9 was being expressed in one of the transformed Microbotryum species; so we did demonstrate a reliable way of implementing CRISPR/Cas9 gene editing in this group of fungi.

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