Our Research

Introduction

Plant immunity involves complex physiological processes that allow plants to recognize, respond to, and defend against various pathogens. These physiological processes encompass a range of interactions and responses at the cellular and molecular levels.

Our Lab is particularly interested in:

-the cellular recognition events by cell-surface localised and intracellular receptor proteins.

-signal transduction pathways connecting receptor-mediated recognition events with downstream (defence) signalling.

-the initiation of cell death and cell wall reinforcement as potent plant host defence strategies.

RECOGNITION - SIGNAL TRANSDUCTION – DEFENCE

Dicots vs. Monocots

Whilst research on model plants provides us with important principles in molecular plant sciences, comparatively little is known about fundamental differences between dicots and monocots.

We make use of the interaction between barley (Hordeum vulgare) and the barley powdery mildew fungus Blumeria hordei tounderstand the physiological plant processes that a plant host-associated microbe manipulates for infection. To unravel overlaps and differences between the physiological processes in monocot and dicot immunity, we also study the interaction between Arabidopsis thaliana and the dicot-infecting mildew fungus Golovinomyces orontii and include other bacterial and fungal microbes that interact with both, mono- and dicotyledonous plants.

Recognition of pathogens

The majority of microbes are not able to infect most plants. To facilitate infection, adapted pathogenic microbes secrete virulence factors (so-called effectors) into the host’s apoplast or directly into host cells (Jones & Dangl, 2006). Effectors target variable host components to promote virulence (Lo Presti et al., 2015; Saur & Hückelhoven, 2020). This ultimately facilitates the pathogen’s reproduction and disease development on susceptible host lines (Fig. 1A).

Figure 1: Schematic representation of the molecular components underlying plant-pathogen interactions. A: Pathogens secret effectors into the plant cytoplasm. Some filamentous microbes (here: Blumeria graminis) rely on specialized feeding structures known as haustoria for effector secretion. Effectors interfere with host components to promote pathogen proliferation and disease development (here: powdery mildew on barley) on susceptible host lines. B: Resistant plants carry resistance genes that often encode intracellular immune receptors (here: MLA). These receptors identify the infection by recognising pathogen effectors (effectors recognised by MLA are known as AVRA effectors). Effector recognition causes a local cell death and resistance. This prevents the spread of obligate biotrophic pathogens, as these pathogens require living host cells for proliferation.

Resistance is often mediated by nucleotide-binding oligomerization domain-like receptors (NLRs). NLRs recognize pathogen virulence effectors inside plant cells, thereby triggering a resistance response that is often associated with a local cell death (Dodds & Rathjen, 2010). Cell death terminates the spread of obligate biotrophic pathogens, as these phytopathogens require living host cells for proliferation (Fig. 1B).
Despite their undisputed importance in the development of diseases, the genetic isolation and functional characterisation of key fungal virulence effectors from the hundreds of small proteins secreted during infection remains challenging (Lo Presti et al., 2015; Uhse & Djamei, 2018). We primarily focus on the powdery mildew AVRA effectors secreted into the barley (Hordeum vulgare) host cells by the powdery mildew fungus Blumeria hordei (Bh). The importance of AVRA effectors in fungal virulence is underlined by the evolutionary concept of host NLRs sensing crucial pathogen effectors: AVRA variants are recognised for resistance to Bh by the allelic barley Mildew locus A encoded NLRs (MLAs) (Mooseman & Schaller 1960; Glawe 2008; Seeholzer et al., 2010; Lu et al., 2016; Maekawa et al., 2018; Saur et al., 2019) (Fig. 1B). Remarkably, the isolate-specific rust effectors AvrSr33 and AvrSr50 encoded by the wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) are recognised by Mla homologs from wheat and rye (Periyannan et al., 2013; Mago et al., 2015; Chen et al., 2017). As such, MLA recognises effectors from entirely unrelated obligate biotrophic phytopathogens. It is thus possibly that the manipulation of AVRA host targets is crucial not only for disease development of powdery mildews, but also for other fungal phytopathogens with a biotrophic lifestyle. As such, we aim is to identify and characterize these manipulations to get insight into the signal transduction pathways involved in plant immunity.

Signal transduction

Pathogen effector functions unravel signal transduction pathways in plant immunity

The NLRs encoded at allelic barley Mla have been studied extensively for decades (Mooseman & Schaller 1960). Only in the last few years, we have been able to isolate the powdery mildew genes encoding the AVRA effectors AVRA1, AVRA6, AVRA7, AVRA9, AVRA10, AVRA13, AVRA22, which are recognised by MLA1, MLA6, MLA7, MLA9, MLA10, MLA13 and MLA22, respectively (Lu et al., 2016, Saur et al., 2019, Bauer et al., 2021). This now lays the ground to molecularly characterize the virulence function of these crucial effectors.

Figure 2: Structural predictions suggest a common fold for the sequence-unrelated AVRA effectors. A: Maximum likelihood phylogeny tree for the 805 predicted secreted proteins of Bgh DH14 lacking respective signal peptides. Isolated AVRA effectors are highlighted; triangles depicting nodes, which are collapsed to allow a more compact visualization. Modified from Saur et al., 2019. B: Structural similarity searches via Intfold v5.0 propose a common fold with a central α-helix facing three to four β-sheets for the powdery mildew AVRA effectors.

The isolated AVRA effectors display at most 8% sequence identity on the amino acid level and we failed to detect any evolutionary conservation (Fig. 2A) between any pair of these proteins (except the effectors encoded at allelic AVRa10/AVRa22) (Saur et al., 2019). This was surprising because the at allelic Mla encoded receptors that recognize the AVRA effectors are over 90% identical on the amino acid level. The big question is therefore: How is it possible that these highly similar receptors detect unrelated effectors? Just recently, updated structural prediction tools were able to shed light into this: Intfold v5.0 was able to detected a common structural fold for the isolated AVRA effectors at the highest confidence level (p<0.0005). Despite the lack of sequence homology, the isolated AVRA effectors are all predicted to harbor one α-helix facing three to four β-sheets (Fig. 2A). This topology is reminiscent of ribonucleases (Fig. 2B).

Figure 3: AVRA effectors lack residues for RNase activity and don’t form a ligand binding pocket. A: Sequence analysis of structural overlay of the Fusarium RNase F1 structure and predicted AVRA13 structure. Amino acids (aa) highlighted in blue form the nucleotide binding pocket in the functional F1 RNase, the once in red highlight the corresponding aa in AVRA13 . Arrows indicate the aa conserved amongst functional RNases and required for enzymatic ribonuclease activity. B: Overlay of the RNase F1 structure (PDB:1FTU) bound to the 2’GMP ligand with the predicted AVRA13 structural fold. Magnification of the RNase F1 ligand binding pocket demonstrates that the respective residues in AVRA13 face away from the 2’GMP ligand bound in the structure of the functional F1 RNase.

In total, ~15% of the Bgh candidate-secreted effector proteins (CSEPs) were predicted to share structural similarities to ribonucleases (Pedersen et al., 2012, Spanu, 2017). Like all these CSEPs, the isolated AVRA effectors lack the conserved catalytic residues for RNases activity (Hill et al., 1983) suggesting that AVRA effectors are unable to process nucleotides (Figure 3A). This catalytic inactivity was confirmed experimentally (Bauer et al., 2021).

Overlay of the functional Fusarium RNase F1 structure (PDB:1FTU) bound to the 2’GMP ligand (Vassylyev et al., 1993) with the predicted AVRA structural folds demonstrates that the AVRA residues that are equivalent to those form the ligand binding pocket in the RNase F1 structure, face away from the 2’GMP ligand of RNase F1 (Fig. 3B, example: AVRA13). The analysis suggests that AVRA effectors lack the ability to process common nucleotide ligands. We have also not detected any ability of heterologous AVRA13 protein to bind and thereby protect ribosomal RNA processing by a functional RNase (Bauer et al. 2021). This leaves us with the question about the virulence function of AVRA effectors and the role of the conserved RNAse-like fold. Because most fungal effectors that are recognized intracellularly by immune receptors function as structural inhibitors of their host targets (Saur et al., 2021), we believe this is also the case for the Bh AVRA effectors.To define this inhibitory or other effector functions, we apply molecular, biochemical and genetic approaches.

Defence

Just getting started here:

We have isolated exciting previously undescribed components that fungal pathogens target for infecting the barley host plant. Importantly, these factors are conserved between monocots and dicots and we are therefore also investigating their function in Arabidopsis thaliana. It appears that the components involve cell wall re-inforcement, which is what we are now investigating using molecular genetic and biochemical approaches. Stay tuned.

Ongoing projects

Here some examples of our ongoing projects:

Isolation of additional AVRA effectors

To enable a broad investigation of the molecular mechanisms underlying AVRA virulence functions (see projects below), we aim to identify further AVRA effectors of Bh by screening a library of RNAse-like CSEPs for their ability to induce MLA-mediated cell death in barley protoplasts (Saur et al., 2019b) and Nicotiana benthamiana (Saur et al., 2019a).

Quantification of virulence function and identification of key residues

Here we quantify the virulence function of the isolated Bh AVRA effectors and their naturally occurring variants that have lost avirulence (AVRA-V). For this, we measure the contribution of the avirulent & of the virulent (AVRA) effectors on the rate of Bh proliferation. We also aim to identify artificial virulence phentoypes by determining if the (AVRA) effectors affect growth of other pathogens. This will clarify whether the avirulence and virulence functions of effectors are dependent/independent of one another and whether the loss of avirulence function is also accompanied by a loss of virulence function.

Biochemical identification of AVRA targets in the barley host

The assignment of biological functions to Bh effectors is of chief importance for understanding barley powdery mildew disease development. We will therefore identifiy AVRA effector targets in barley host cells. We apply conventional immunoprecipitation approaches to isolate specific host targets of individual AVRA effectors and analyze their connectivity. Using the same transgenic AVRA expressing plants, we apply state-of-the-art proximity-dependent protein labeling techniques to identify the in situ interaction characteristics of AVRA effectors to facilitate the identification of the biochemical process in which AVRA host targets participate.

Identification of the host cell death pathways affected in the presence of Bh AVRA effectors

As an obligate biotroph, Bh relies on living host tissue for proliferation. The inhibition of host cell death is therefore of utmost importance for the proliferation of Bh (Saur et al., 2021, Saur & Hückelhoven 2021). Because the MLAs recognise effectors from multiple unrelated pathogens with biotrophic lifestyles, the by MLA-recognised AVRA effectors are strong candidates for directly and/or indirectly inhibiting barley cell death to allow Bh proliferation. To test this, we investigate the suppressive effect of the AVRA and related virulence effectors on a variety of cell death pathways in barley.

How does Blumeria hordei and its manipulations of the barley host plant affect colonisation by other microbes

In this project we study how Bh affects the barley colonisation by other microbes, both directly by microbial antagonisms and indirectly by induces physiological changes of the barley host plant. We focus on microbes of the barley microbiota and dominant invaders with both, biotrophic and necrotrophic lifestyles.

Literature

Bauer S, Yu D, Lawson AW, Saur IML, Frantzeskakis L, Kracher B, Logemann E, Chai J, Maekawa T, Schulze-Lefert P. 2021. The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold. PLoS Pathogens 17(2):e1009223.

Chen J, Upadhyaya NM, Ortiz D, Sperschneider J, Li F, Bouton C, Breen S, Dong C, Xu B, Zhang X, Mago R, Newell K, Xia X, Bernoux M, Taylor JM, Steffenson B, Jin Y, Zhang P, Kanyuka K, Figueroa M, Ellis JG, Park RF, Dodds PN. 2017. Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat. Science 358(6370): 1607-1610.

Dodds PN, Rathjen JP. 2010. Plant immunity: towards an integrated view of plant-pathogen interactions. Nature Reviews Genetics 11(8): 539-548.

Hill, C.P., Dodson, G.G., Heineman, U., Saenger, W., Mitsui, Y., Nacamura, K., Borisov, S., Tishenko, G., Polyakov, K., and Pavlovsky, S. 1983. The structural and sequence homology of a family of microbial ribonucleases. Trends in Biochemical Sciences 8:364–369.

Lo Presti L, Lanver D, Schweizer G, Tanaka S, Liang L, Tollot M, Zuccaro A, Reissmann S, Kahmann R. 2015. Fungal Effectors and Plant Susceptibility. Annual Review of Plant Biology 66: 513-545.

Lu XL, Kracher B, Saur IML, Bauer S, Ellwood SR, Wise R, Yaeno T, Maekawa T, Schulze-Lefert P. 2016. Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen. Proceedings of the National Academy of Sciences of the United States of America 113(42):E6486-E6495.

Maekawa T, Kracher B, Saur IML, Yoshikawa-Maekawa M, Kellner R, Pankin A, von Korff M, Schulze-Lefert P. 2018. Subfamily-Specific Specialization of RGH1/MLA Immune Receptors in Wild Barley. Molecular Plant Microbe Interactions 32(1):107-119.

Mago R, Zhang P, Vautrin S, Simkova H, Bansal U, Luo MC, Rouse M, Karaoglu H, Periyannan S, Kolmer J, et al. 2015. The wheat Sr50 gene reveals rich diversity at a cereal disease resistance locus. Nature Plants 1(12):5118. Moseman JG, Schaller CW. 1960. Genetics of the Allelic Series at the Mla Locus in Barley and Cultures of Erysiphe-Graminis F Sp Hordei That Differentiate These Alleles. Phytopathology 50(10): 736-741.

Pedersen C, Ver Loren van Themaat E, McGuffin LJ, Abbott JC, Burgis TA, Barton G, Bindschedler LV, Lu X, Maekawa T, Wessling R, Cramer R, Thordal-Christensen H, Panstruga R, Spanu PD. 2012. Structure and evolution of barley powdery mildew effector candidates. Bmc Genomics 13:694

Periyannan S, Moore J, Ayliffe M, Bansal U, Wang X, Huang L, Deal K, Luo M, Kong X, Bariana H, Mago R, McIntosh R, Dodds P, Dvorak J, Lagudah E. 2013. The Gene Sr33, an Ortholog of Barley Mla Genes, Encodes Resistance to Wheat Stem Rust Race Ug99. Science 341(6147):786-788.

Saur IML, Panstruga R, Schulze-Lefert P. 2021. NOD-like receptor-mediated plant immunity: from structure to cell death. Nature Reviews Immunology 21(5):305-318.

Saur IML, Hückelhoven R. 2020. Humboldt Review: Recognition and defence of plant-infecting fungal pathogens. Journal of Plant Physiology 256(153324).

Saur IML, Bauer S, Kracher B, Lu XL, Franzeskakis L, Muller MC, Sabelleck B, Kummel F, Panstruga R, Maekawa T, et al. 2019a. Multiple pairs of allelic MLA immune receptor-powdery mildew AVR(A) effectors argue for a direct recognition mechanism. Elife 8:e44471

Saur IML, Bauer S, Lu XL, Schulze-Lefert P. 2019b. A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors. Plant Methods. doi: 10.1186/s13007-019-0502-0.

Seeholzer S, Tsuchimatsu T, Jordan T, Bieri S, Pajonk S, Yang WX, Jahoor A, Shimizu KK, Keller B, Schulze-Lefert P. 2010. Diversity at the Mla Powdery Mildew Resistance Locus from Cultivated Barley Reveals Sites of Positive Selection. Molecular Plant-Microbe Interactions 23(4):497-509.

Spanu PD. 2017 Cereal immunity against powdery mildews targets RNase-Like Proteins associated with Haustoria (RALPH) effectors evolved from a common ancestral gene. New Phytologist 213:969–971.

Uhse S, Djamei A. 2018. Effectors of plant-colonizing fungi and beyond. Plos Pathogens 14(6):e1006992

Vassylyev DG, Katayanagi K, Ishikawa K, Tsujimoto-Hirano M, Danno M, Pähler A, Matsumoto O, Matsushima M, Yoshida H, Morikawa K. 1993 Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2’GMP. Journal of Molecular Biology 230(3):979-96.

© Isabel Saur