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De- and reconstructing virulence strategies of fungal plant pathogens

Periodic Reporting for period 3 - conVIRgens (De- and reconstructing virulence strategies of fungal plant pathogens)

Okres sprawozdawczy: 2021-06-01 do 2022-11-30

Pathogenic fungi are an increasing threat to plants and animals, causing significant losses in worldwide food production. Prominent examples of fungal pathogens causing devastating crop damage are soybean rust (Phakopsora pachyrhizi), wheat stem rust (Puccinia graminis f. sp. tritici) and wheat blast (Magnaporthe oryzae. Many important plant diseases are caused by biotrophic pathogens, which colonize and feed from living host tissue. The largest groups of fungal biotrophs are the rust fungi, the powdery mildews, and the smut fungi. The biotrophic virulence strategy requires efficient suppression of immune responses and reprogramming of host metabolism to feed the pathogen. To manipulate the host and promote infection, pathogens secrete large repertoires of virulence proteins, collectively termed “effectors“. Efficient plant immunity can be induced upon recognition of effectors by specific resistance (R) proteins, reflecting the evolutionary arms race between plants and pathogenic microbes.
The smut fungi (Ustilaginomycota) comprise >1200 species, infecting approximately 4000 plant species, including all economically relevant monocot crops. All Ustilaginales smuts are strictly biotrophic parasites, but unlike obligate biotrophs, their life cycle involves a saprophytic phase represented by a haploid yeast stage (sporidia). On host plants, compatible sporidia recognize each other via a pheromone-receptor system and fuse to form dikaryotic filaments; it is these filaments that initiate the pathogenic fungal development.
Genome sequences of seven plant pathogenic Ustilaginales species have been published. All these genomes are small (about 20 Mbp), encode about ~7.000 proteins and show rather low content of repetitive elements and gene duplications. Together, these features reinforce the excellent suitability of these species for functional genetic and genomic approaches.
This project aims to utilize the excellent genetic accessibility of the smut model fungus Ustilago maydis to approach a previously impossible, pioneering enterprise: the synthetic reconstruction of eukaryotic plant pathogens. In a first step, fungal virulence will be deconstructed by consecutive deletion of the U. maydis effector repertoire to generate disarmed mutants. These strains will serve as chassis for subsequent reconstruction of fungal pathogenicity from different sources. A combination of transcriptomics and comparative genomics will help to define synthetic effector modules to reconstruct virulence in the chassis strains.
Deconstruction of U. maydis virulence will identify a complete arsenal of fungal virulence factors. Reconstruction of virulence will show how effector modules determine fungal virulence, including those of the previously not accessible obligate biotrophs. conVIRgens will thereby provide fundamentally new insights and novel functional tools towards the understanding of microbial virulence.
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).
Current progress includes methods improvement beyond state of the art at beginning of the project. This includes:
- setup of high fidelity CRISPR-Cas9 mutagenesis in U. maydis
- setup of CRISPR-Cas9- mediated, marker- and scar less gene replacement in U. maydis
- construction of artificial effector gene clusters using modular cloning strategy
- heterologous expression of fungal virulence factors in U. hordei

We have performed:
- comparative transcriptomics of U. maydis infecting different maize lines
- comparative transcriptomics of U. maydis and S. reilianum
- genome sequencing U. maydis and U. hordei field isolates
- Pac-bio de-novo sequencing of three smut species

Resulting in the following key findings:
- identification of maize-line specific activity of effector genes (Schurack et al., 2021)
- identification of tumor-related effectors in U. maydis in comparison with S. reilianum (Zuo et al., 2020)

Until end of the project, we aim to complete the major goals of the project. In particular we aim to:
- de- and reconstruct the effector repertoire responsible for U. maydis induced formation of plant tumors
- dissect the genetic basis of host jump / host adaptation in smut pathogens
- functionally express and characterize effectors of rust fungi in U. maydis