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.​​

We’re looking forward to the new year with four new virtual seminars! This series is freely available to all.

Abstracts for the ePosters prese​nted during the July 12–13: Molecular Mechanism and Structure—Zooming in on Plant Immunity and September 15–16: Pathogen-Host Coevolution—Combating Resistance Breaking in Agriculture e-Symposia have been posted to the July and September issues of MPMI. This is a great opportunity for those who were unable to attend the eSymposia to take in the emerging science presented at this year’s online meeting.​

Associate professor of molecular virology at the
University of Nebraska-Lincoln Hernán García-Ruiz used the Top 10 Unanswered Questions in MPMI​ to inspire the students in his introductory graduate-level course. Learn more about his process in the interview below.

A group of nonprofit plant science organizations and societies have assembled to Root Out Oppression Together and Share Our Outcomes Transparently (ROOT & SHOOT), through a Research Coordination Network Grant funded by the National Science Foundation. Our overarching goal is to transform our discipline by getting to the root of the systemic oppressions that exist within our organizations (and ourselves) so we can regrow as inclusive, liberated, spaces.

We are seeking an organization, individual, or set of individuals (partnerships) who will work with us to achieve our goal. We need help to cocreate equitable and inclusive practices for our community; provide sustainable, inclusive, equitable, anti-racist/anti-oppression leadership training; guide power and equity assessments within our participating organizations; and consult for intervention programs. More detail on these projects can be found here.

We will start reviewing applications on January 14, 2022. We aim to start working with successful applicant(s) in April 2022.

September

The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 during Inception of Symbiosis in Arachis hypogaea

Pritha Ganguly, Dipan Roy, Troyee Das, Anindya Kundu, Fabienne Cartieaux, Zhumur Ghosh, and Maitrayee DasGupta

The long noncoding RNA ENOD40 is required for cortical cell division during root nodule symbiosis (RNS) of legumes, although it is not essential for actinorhizal RNS. Ganguly et al. set out to understand whether ENOD40 is required for aeschynomenoid nodule formation in Arachis hypogaea. AhENOD40 expresses from chromosomes 5 (AhENOD40-1) and 15 (AhENOD40-2) during symbiosis, and RNA interference by these transcripts drastically affects nodulation, indicating the importance of ENOD40 in A. hypogaea.

​Metatranscriptomic Comparison of Endophytic and Pathogenic Fusarium–Arabidopsis Interactions Reveals Plant Transcriptional Plasticity​

Li Guo, Houlin Yu, Bo Wang, Kathryn Vescio, Gregory A. DeIulio, He Yang, Andrew Berg, Lili Zhang, Véronique Edel-Hermann, Christian Steinberg, H. Corby Kistler, and Li-Jun Ma​

Two strains of the fungus Fusarium oxysporum (Fo) share a core genome, but one is a beneficial endophyte while the other is a detrimental pathogen causing wilt and death. Guo et al. ​tried to tease apart why these two strains cause such opposite reactions, and more generally how plants respond differently to useful and harmful microbes, by exploring the interaction of these two strains with the model plant Arabidopsis.

October

Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling during Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne

Nazanin Noorifar, Matthew S. Savoian, Arvina Ram, Yonathan Lukito, Berit Hassing, Tobias W. Weikert, Bruno M. Moerschbacher, and Barry Scott

A diverse set of microbes survives and thrives inside plants as endophytes, but we have little mechanistic understanding of these intimate associations. In their study, Noorifar et al. show the way in which an Epichloë endophyte remodels its cell wall, converting chitin to chitosan, to avoid detection by host defenses. Deletion mutants reveal an important role for chitin deacetylases in hyphal growth inside the plant.

November

Computational Structural Genomics Unravels Common Folds and Novel Families in the Secretome of Fungal Phytopathogen Magnaporthe oryzae

Kyungyong Seong and Ksenia V. Krasileva

Recent breakthroughs in protein structure modeling demonstrate the ability to predict protein folds without depending on homologous templates. In their study, Seong and Krasileva employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, they predicted the folds of 1,295 proteins (70%).

We’re looking forward to the new year with four new virtual seminars! This series is freely available to all.