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 graminis forma specialis hordei (Bgh). 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 Bgh 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. Our group’s aim is to identify and characterize these manipulations

AVRA function

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 . Errors 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 don’t only lack the ability to process nucleotides, but are also unable to bind nucleotide ligands. In agreement, 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.

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 Bgh AVRA effectors.

To define this inhibitory or other effector functions, we apply unbiased molecular, biochemical and genetic approaches.


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© Isabel Saur