A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanisation Pathway

Name: Yan Xu

Current Position: Botany, University of British Columbia, Vancouver, BC, Canada

Brief Bio: I am excited to have our paper, "A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanisation Pathway," published in MPMI. This project was initiated by me four years ago when I became a Ph.D. student in Dr. Xin Li's lab at the University of British Columbia. The goal of my Ph.D. thesis was to explore the development and pathogenesis of a notorious, but understudied, phytopathogen, S. sclerotiorum.

If you visit our lab's website, you will find that we mainly study the molecular mechanisms of plant innate immunity using the model plant Arabidopsis thaliana. We are basically a plant lab without any other lab members who had previous experience with this pathogen, except for my supervisor, who studied S. minor during her Ph.D. program. You can image how hard it was and how many setbacks I have encountered during my research.

The first obstacle I had was how to obtain mutants with phenotypes of interest. Forward genetic approaches are often utilized to screen for mutants after random mutagenesis. For most fungal research, asexual conidia are used to conduct genetic screens. However, this fungus does not produce conidia. Meanwhile, the multinucleate feature of its asexual tissues rendered the problem worse. After several failed attempts with mutagenizing sclerotia, we ended up using sexual, haploid ascospores, which turned out to be ideal for mutagenesis. The next question was selection of a suitable mutagen. We first tried EMS, which is broadly applied in Arabidopsis studies. However, this chemical was problematic, because it killed all ascospores after mutagenesis and washes. Finally, we settled on a relatively mild mutagen, UV irradiation, and were able to acquire many mutants with the desired phenotypic defects.

Next, we sequenced many of our mutants using next generation sequencing (NGS) since the expense of NGS has decreased drastically over the past few years. After analyzing the NGS data, I was able to find several candidates for each of my mutants. The biggest problem I had at the time was to knock out the candidate genes to determine which in the mutation is responsible for my mutant phenotype. Targeted gene replacement by homologous recombination has been applied in many fungi with relatively high efficiency. However, this method did not help me obtain any knockout mutants during six months of attempts. After modifying the protocol many times using modifications from relevant literature, we ultimately set up our own protocol for successful targeted gene disruption.

Looking back, with every step I moved forward, I encountered unpredicted difficulties. Although sometimes frustrating, I really enjoyed identifying the problems and solving them. I hope that the forward genetic pipeline mentioned in this paper can be applied to facilitate in-depth studies of other nonmodel fungal species in the future.

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