In the first project phase, both the major parts of the project could have been initiated.
Prerequisite of the deconstruction approach is the efficient gene editing in U. maydis using CRISPR/Cas9. In this regard, an optimized protocol has been established for U. maydis, using a modified Cas9 version (Cas9HF). Also, by full genome sequencing of a set of transformants we could demonstrate that gene disruption using this tool does not lead to the generation of off-target mutations, which is an important step for the planned approach. The results have been summarized in a manuscript, which has been recently published (Zuo et al., 2020).
We performed transcriptome analysis of U. maydis infecting different maize lines to elucidate fine-tuned host adaptation. This approach identified maize-line specific activity of U. maydis effectors, providing novel insight in the co-evolution of the pathogen with its host (Schurack et al., 2021). To further dissect tumor-related effectors and better understand life-style differentiation between U. maydis and its close relative Sporisorium reilianum, comparative transcriptome profiling of both organisms has been performed. This identified a set of effectors which are specifically induced in U. maydis during tumor induction in comparison to their respective one-to-one homologs in S. reilianum. Gene deletion together with CRISPR-Cas9-based gene replacement revealed that both transcriptional regulation and sequence diversification of effector proteins contribute to species-specific functionalization (Zuo et al., 2020).
To reconstruct fungal virulence, two complementary approaches are followed: a) the computational approach which is mainly aiming on comparative genomics-based characterization of effector repertoires, and b) the experimental approach to generate artificial effector gene clusters and to perform gain of function genetic experiments by expressing heterologous effectors in U. maydis.
We have performed de-novo sequencing of smut fungi has been performed using PacBio and Nanopore sequencing. We generated annotated de-novo genomes for three fungal species. In addition, we investigated intraspecific variation by sequencing several Ustilago hordei and Ustilago maydis strains which have been isolated around the globe. Sequencing of Chinese and European U. maydis strains was integrated in an evolutionary analysis of effectors within U. maydis, as well as in comparison with S. reilianum. This approach could identify a link between expression pattern and sequence variation / evolutionary speed of effector genes (Depotter et al., 2020). Complementary to this, we established tools in the barley smut U. hordei to express heterologous virulence factors, which also allows to study effectors of obligate biotrophs pathogens of barley (Ökmen et al., 2021).
We successfully generated annotated genomes for Ustilago striiformis, Ustilago nuda, and Ustilago tritici. Additionally, we explored intraspecific variation by sequencing six Ustilago hordei strains and 14 Ustilago maydis strains collected from various locations worldwide. Our findings revealed that the invasion of transposable elements into smut genomes has led to an increased rate of nucleotide substitution, driving effector evolution. This process likely contributes to host jumps and the adaptation of smut pathogens. Manuscripts detailing these results have been published in 2021 and 2022 (Depotter et al., 2021, 2022).
To achieve a more efficient genetic modification of S. reilianum, we established a CRISPR-Cas9 based transformation protocol for this organism. Here, a ribonucleoprotein (RNP)-mediated CRISPR/Cas9 method for mutagenesis in S. reilianum was developed. We evaluated the efficiency of this approach through both in vitro cleavage assays and in vivo experiments using a GFP-expressing S. reilianum strain. We successfully applied this method to generate frameshift and knock-out mutants in S. reilianum without introducing a resistance marker, instead utilizing an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 technique demonstrated enhanced mutagenesis efficiency and versatility, being applicable to various types of mutations. Importantly, this approach enables marker-free genome editing in S. reilianum, representing a significant advancement in fungal genetic manipulation techniques (Werner et al., 2024).
