Powdery mildews are fungal diseases that affect many plants, including important crops such as barley. The fungi behind these diseases deliver molecules known as effectors inside plant cells, which manipulate the plants' biology and help the fungus to invade the plants' tissues. In response, some plants have evolved immune receptors encoded by so-called R genes (short for resistance genes) that detect the effectors inside the plant cell and trigger an immune response. The response often kills the plant cell and those nearby to limit the spread of the fungus. Effectors that are recognized by host immune receptors are termed avirulence effectors (or AVRs for short).

Scientists tend to assume that most effectors do not bind directly to their immune receptors. Instead, it is thought that the immune receptors are more likely to be detecting a change in some other plant protein that is caused by the effectors' activities.

In barley populations, one R gene that protects against powdery mildew encodes an immune receptor known as MLA. Different plants can carry subtly different versions of this R gene meaning that they make similar but different variants of the same receptor. Each MLA variant confers immunity only to strains of powdery mildew that carry the matching AVR effector. A few AVR effectors from powdery mildews have been identified, but most AVR effectors from powdery mildews remain unknown.

Saur et al. looked for new AVR effectors from powdery mildew fungi collected in the field, and found four that were recognized by barley plants carrying MLA variants. Two of these new effectors were fairly similar to each other, but they were all unlike those that had been identified previously.

When Saur et al. engineered barley cells to make these new AVRs alongside their matching MLA receptors, the cells died – which is consistent with the expected immune response. Similar experiments with distantly related tobacco plants agave the same results. This suggested that the immune receptors did not need any other barley proteins to recognize the effectors, indicating that the interaction between the two may be direct. Indeed, two other techniques that test for direct protein-protein interactions, – one that involved extracts from tobacco leaves, and another that involved yeast, – gave results consistent with a direct interaction between the MLA receptor variants and the fungal effectors.

Plant disease is still a major cause of loss of yield in crops. Transferring an R gene from one plant species to another is a potentially powerful approach to help crops resist disease. The discovery that multiple variants of the same resistance gene can bind to dissimilar effectors from a disease-causing fungus in distantly related plant species underlines the potential of this approach.

The NLR family of immune receptors is structurally conserved between animals and plants, perceives non-self and modified-self molecules inside host cells, and mounts potent innate immune responses to terminate microbial pathogenesis (Maekawa et al., 2011a). Animal NLRs are normally activated by conserved microbe- or damage-associated molecular patterns (MAMPs/DAMPs), whereas plant NLRs typically detect strain-specific pathogen effectors, designated avirulence effector proteins (AVRs) (Maekawa et al., 2011a; Jones et al., 2016). Plant NLRs recognize either the effector structure or sense effector-mediated modifications of host proteins (Jones et al., 2016). Single plant NLRs or NLR pairs can also detect the manipulation of effector target mimics, called decoys, and a variation of the latter mechanism involves the direct integration of decoy domains mimicking host targets within NLRs, termed integrated decoy domains (Jones et al., 2016; Cesari et al., 2014; Kroj et al., 2016). The selective forces shaping the evolution of the comparatively small complement of NLRs in vertebrates are incompletely understood (~20 family members, (Lange et al., 2011; Meunier and Broz, 2017)), but in plant species co-evolution with host-adapted pathogens has strongly influenced the expansion and diversification of NLR repertoires (Jacob et al., 2013; Meyers et al., 2005).

Plant disease resistance (R) genes to host-adapted pathogens often encode NLRs, are frequently members of large gene families organized in complex clusters of paralogous genes, and can rapidly evolve through a range of natural gene diversification mechanisms (Jacob et al., 2013; Meyers et al., 2005). There are several examples of allelic series of NLR-type R genes known in plants (Ellis et al., 1999; Allen et al., 2004; Srichumpa et al., 2005; Seeholzer et al., 2010; Kanzaki et al., 2012). In these cases, multiple distinct recognition specificities evolved in the host population at a single R gene with each allele detecting a corresponding strain-specific AVR in the pathogen population. Such multi-allelic NLR-type R genes are particularly instructive for studying the underlying co-evolutionary process between host and pathogen.

Ascomycete powdery mildews are widespread pathogens of thousands of angiosperm plant species in temperate climates, including economically relevant crops (Glawe, 2008). They are obligate biotrophic pathogens, meaning that their growth and reproduction is entirely dependent on living host cells. The haploid barley powdery mildew pathogen Blumeria graminis forma specialis hordei (Bgh) multiplies mainly clonally and is a member of the species Blumeria graminis that is specialized for its host plant barley (Hordeum vulgare). There are various specialized forms (formae speciales or fs. spp.) of B. graminis, each of which is capable of infecting the respective host plant species belonging to the grass (Poaceae) family, including cereals such as barley and wheat (Wyand and Brown, 2003). Within each forma specialis, numerous isolates (strains) can be distinguished in the pathogen population, based on their respective virulence or avirulence infection phenotypes vis-à-vis particular genotypes of the host population (Lu et al., 2016). The genomes of powdery mildews are characterized by the loss of several, otherwise widely conserved Ascomycete genes with functions related to carbohydrate degradation and primary and secondary metabolism (Spanu et al., 2010; Wicker et al., 2013), and this is believed to explain their strict dependence on living plant cells. Similar to other filamentous phytopathogens, grass-infecting powdery mildew genomes harbor hundreds of candidate secreted effector protein (CSEP)-coding genes, which are assumed to contribute to fungal pathogenesis (Wicker et al., 2013; Pedersen et al., 2012; Guttman et al., 2014). Pathogen effectors often work by subverting innate immune responses, thereby facilitating host colonization and disease (Rovenich et al., 2014).

Domesticated barley and wheat contain numerous powdery mildew R gene loci that were often introgressed from their corresponding wild relatives (Jørgensen and Wolfe, 1994; Lutz et al., 1995; Maekawa et al., 2019). In both barley and its close relative wheat, one of these powdery mildew R loci, designated mildew locus a (Mla) and powdery mildew 3 (Pm3), respectively, has been subject to exceptional functional diversification, resulting in large numbers of Mla or Pm3 recognition specificities (Seeholzer et al., 2010; Lutz et al., 1995; Bhullar et al., 2010). Although wheat Pm3 and barley Mla loci each span a cluster of NLR genes, known Pm3 and Mla recognition specificities to the B. graminis pathogen appear to have arisen from allelic diversification of a single NLR gene in the corresponding NLR clusters (Seeholzer et al., 2010; Maekawa et al., 2019; Shen, 2003). Isolate-specific disease resistance to Bgh mediated by MLA receptors is invariably linked to the activation of localized host cell death, and this immune response likely terminates growth of the biotrophic pathogen by shutting off its nutrient supply (Boyd et al., 1995). Of note, the Mla orthologs Sr33 in wheat (Periyannan et al., 2013) and Sr50 in rye (Mago et al., 2015) confer disease resistance to the stem rust pathogen Puccinia graminis f. sp. tritici (Pgt) isolate Ug99, a major threat to global wheat production. Pgt and Bgh belong to the Basidiomycota and Ascomycota phyla, respectively, indicating that MLA receptors can detect the presence of independently evolved avirulence effectors. Barley Mla and wheat Pm3 both encode intracellular NLRs with an N-terminal coiled-coil (CC) domain but lack significant sequence relatedness (Zhou et al., 2001), whereas 23 allelic barley MLA resistance proteins exhibit >91% amino acid (aa) sequence identity (Seeholzer et al., 2010), and 17 deduced allelic wheat Pm3 receptors share >97% aa sequence identity (Bhullar et al., 2010). Diversifying selection among resistance alleles of Mla and Pm3 is largely confined to regions encoding the C-terminal leucine-rich repeats (LRR) of the receptors (Seeholzer et al., 2010; Maekawa et al., 2019; Bhullar et al., 2010). The polymorphic MLA LRR is critical for effector detection as shown by a series of reciprocal domain swaps between MLA1 and MLA6 (Shen, 2003). Barley Mla1 confers race-specific disease resistance to a Bgh isolate carrying the cognate avirulence gene AVR_a1 in transgenic Arabidopsis thaliana, suggesting ∼ 150 million years of evolutionary conservation of the underlying immune mechanism and potentially pointing to a direct recognition mechanism of AVR_A effectors that is highly conserved between barley and A. thaliana (Maekawa et al., 2012).

Recently, the Bgt avirulence gene AvrPm3^a2/f2, recognized by the wheat Pm3a and Pm3f alleles, was identified by a map-based cloning approach and found to encode a typical CSEP that belongs to an effector family of 24 members (Bourras et al., 2015). The clonal nature of the haploid Bgh pathogen facilitated the identification of isolate-specific sequence variation in the transcriptomes of a global collection of 17 Bgh strains. These were associated with Bgh pathotypes on Mla1- and Mla13-containing near-isogenic barley lines to identify sequence-unrelated AVR_a1- and AVR_a13-encoding CSEPs (Lu et al., 2016). However, it remains unclear whether these powdery mildew avirulence effectors bind directly to the corresponding NLR receptors or whether the receptors sense the presence of the pathogen indirectly through effector-mediated modifications of host proteins.

To reveal the molecular mechanism underlying the co-evolutionary diversification of barley MLA NLRs with host-adapted Bgh, we first described the natural AVR_a gene diversity in a local Bgh population. This allowed us to isolate four additional AVR_A effectors by applying a transcriptome-wide association study (TWAS) with high-confidence single nucleotide polymorphisms (SNPs) underlying disease resistance/susceptibility phenotypes in barley. We then employed co-transfection experiments to show that only matching Mla-AVR_a pairs activate cell death in barley leaf protoplasts. Agrobacterium tumefaciens-mediated co-delivery of Mla-AVR_a pairs in Nicotiana benthamiana leaves demonstrated that their co-expression is sufficient to trigger cell death in this heterologous plant species. The AVR_a avirulence effectors encode CSEPs that are sequence-unrelated except for allelic AVR_A10 and AVR_A22. These findings are inconsistent with a co-evolutionary arms race model describing iterative cycles of MLA receptor and AVR_A effector adaptations. Moreover, we failed to detect cell death activity for the previously reported EKA_AVR_a10, a member of the EKA gene family that is derived from part of a class-1 LINE retrotransposon (Ridout, 2006; Amselem et al., 2015). Using co-expression experiments with matching Mla-AVR_a pairs, including previously reported AVR_a13, we present evidence for direct receptor-avirulence effector interactions. Our findings imply that MLA receptors have an exceptional propensity to directly detect sequence-unrelated and likely structurally diverse pathogen effectors and that this feature might have facilitated the functional diversification of the receptor in the host population.

Previous pathotyping studies of Bgh field isolates with barley varieties carrying different powdery mildew R genes suggested that the Central European pathogen population can be considered as a single epidemiological unit (Limpert, 1987). We have shown here that among 13 Bgh isolates from a local population in Germany ten virulence combinations can be distinguished in interactions with a panel of Mla NILs, suggesting potential adaptation of the pathogen population to multiple Mla resistance specificities. Our findings are congruent with a recent study describing a high complexity of Central European Bgh virulence pathotypes on a panel of 50 differential barley varieties carrying Mla or other powdery mildew R genes (Dreiseitl, 2014). The same study also provided evidence for a complete separation of the Central European and Australian Bgh populations with non-overlapping pathotypes and estimated an almost three-fold higher virulence complexity for the European population. This is believed to be due to the cultivation of barley varieties carrying distinct powdery mildew R gene combinations on the two continents, which leads to differential intensities of directed selection on Bgh populations. Analysis of our isolate collection comprising, among others, 13 newly characterized Bgh strains from a local population in Germany and three representative Australian isolates, is supportive of a significantly greater virulence complexity of the Central European over the Australian isolates even when considering virulence patterns only on Mla NILs (Supplementary file 1). The complexity of avirulence and virulence alleles in the Central European population suggests that the fungus adapts by balancing selection of AVR_a genes among strains rather than selective sweeps to maintain pathogenicity.

Specific activation of cell death upon transient gene expression of matching AVR_a and Mla gene pairs in barley protoplasts and heterologous N. benthamiana provided evidence for the function of candidate AVR_a7, AVR_a9, AVR_a10, and AVR_a22 as avirulence effectors, all of which encode Bgh CSEPs with a predicted signal peptide. This is different from the previously reported EKA_AVR_A10, which lacks a canonical signal peptide, belongs to the large EKA gene family that is derived from part of a class-1 LINE retrotransposon (Ridout, 2006; Amselem et al., 2015), and was used as an elicitor of induced MLA10 nuclear interaction with a WRKY transcription factor (Shen et al., 2007). However, we were unable to detect MLA10-mediated cell death activity for EKA_AVR_A10. Our findings demonstrate that virulent Bgh isolates escape recognition of corresponding MLA receptors predominantly by non-synonymous SNPs but also loss of expression of the corresponding genes (Figure 3A; Lu et al., 2016). Upon in silico removal of the signal peptide, phylogenetic analysis for the 805 predicted secreted proteins of Bgh (Frantzeskakis et al., 2018) and comparative sequence analysis of AVR_a7, AVR_a9, AVR_a10, AVR_a22, and previously reported AVR_a1 and AVR_a13, also representing CSEPs, failed to detect evolutionary conservation (Figure 3—figure supplement 1) or significant sequence similarity (at most 8% sequence identity) between any pair of these polypeptides except for allelic AVR_a10 and AVR_a22 (Figure 3C and E). By contrast, MLA7, MLA9, MLA10 and MLA13 receptors are >96% identical in aa sequence to each other, whereas MLA1 and MLA22 are more diverged and share 91% identical polypeptide sequences with this receptor group (Seeholzer et al., 2010). This raises questions regarding the evolutionary history of MLA10- AVR_A10 and MLA22-AVR_A22 receptor-effector pairs. AVR_a10 and AVR_a22 are notable for several reasons: (i) these allelic effectors define two dominant haplotypes of CSEP0141 in Bgh, (ii) CSEP0141 belongs to a core of 190 effectors that are conserved among different B. graminis f. sp. and, therefore, likely contributes to pathogen fitness, (iii) CSEP0141 represents the core effector with the highest frequency of non-synonymous SNPs among the tested global collection of Bgh isolates, and (iv) a single dominant haplotype of its Bgt ortholog, designated BgtE-5921, was found in the wheat powdery mildew population. Collectively, this suggests a model in which the two dominant CSEP0141 haplotypes, AVR_a10 and AVR_a22, evolved in Bgh by the action of two opposing selective forces: functional conservation that maintains pathogen fitness through retention of effector virulence activity and sequence diversification to escape recognition by MLA10 and MLA22 receptors, respectively. AVR_a10 and AVR_a22 likely represent a balanced polymorphism in the extant pathogen population because Bgh isolates containing one or the other haplotype do not form discrete subgroups (Figure 1—figure supplement 1A). However, the likely source of many, if not all, Mla recognition specificities in domesticated barley is wild barley, H. spontaneum (Jørgensen and Wolfe, 1994). Thus, it is possible that Mla10, Mla22 and their matching avirulence effector genes have not diversified during a co-evolutionary arms race (Ravensdale et al., 2011) but have rather evolved independently in separate host and pathogen populations. In such a scenario, the balanced AVR_A10 and AVR_A22 polymorphism in the extant pathogen population is the consequence of concurrent cultivation of domesticated barley varieties with these Mla resistance specificities. Besides an apparently balanced AVR_a10 and AVR_a22 polymorphism at CSEP0141 in the Bgh field population one would expect sporadic strains that are virulent on both Mla10- and Mla22-harboring host varieties, which is the case for Bgh isolate DH14 carrying a SNP that introduces a single aa substitution in AVR_a10 (Figure 3). In such strains, a fitness penalty for the pathogen might be mitigated by residual virulence activity of the CSEP0141 variant.

Our split-LUC and yeast two-hybrid experiments provided evidence for direct and specific interactions between multiple matching MLA/AVR_A pairs (Figure 5). More than 50 years after the original discovery of multi-allelic race-specific disease resistance at Mla (Moseman and Schaller, 1960), these findings now imply that the co-evolutionary functional diversification of these immune receptors is at least in part mechanistically underpinned, and was perhaps driven by direct interactions with sequence-unrelated Bgh avirulence effectors. Direct receptor-avirulence effector interactions have been described for flax L and rice Pik multi-allelic disease resistance genes, which encode NLR-type receptors, (Kanzaki et al., 2012; Dodds et al., 2006). In flax, a subset of allelic L receptors (L5, L6, and L7) interact with a specific subset of highly sequence-related flax rust AvrL567 proteins, and in rice, allelic Pik receptors interact with specific variants of highly sequence-related AVR-Pik proteins. Rice Pik immune receptors contain an integrated heavy metal-associated RATX1/HMA domain, which binds directly to AVR-Pik and Pik functional diversification is driven by polymorphisms in this integrated domain (Kanzaki et al., 2012; Maqbool et al., 2015; De la Concepcion et al., 2018). Barley MLA and flax L proteins lack detectable integrated domains, and diversifying selection in allelic MLA receptors is largely confined to predicted solvent-exposed residues of leucine-rich repeats 7 to 15 (Seeholzer et al., 2010). Thus, to the best of our knowledge, a co-evolutionary functional diversification of multi-allelic NLR-type receptors in plants with directly recognized sequence-unrelated avirulence effectors, as described here for matching MLA, is without precedence. Effectors AVR1-CO39 and AVR-Pia of the ascomycete Magnaporthe oryzae are sequence-unrelated but have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge and are both recognized by the rice NLR pair RGA4-RGA5 through the integrated RATX1/HMA domain located at the C-terminus of RGA5 (de Guillen et al., 2015; Cesari et al., 2013). Structural similarity searches then showed that AVR1-CO39 and AVR-Pia are founders of a family of sequence-unrelated but structurally conserved fungal effectors in a broad range of ascomycete phytopathogens (de Guillen et al., 2015). Consistent with structural modelling (IntFOLD Version 3.0 (McGuffin et al., 2015)), the recently resolved NMR-based and crystal structure of Bgh CSEP0064 revealed a ribonuclease-like fold (Pennington et al., 2019) with the absence of canonical catalytic residues in the substrate-binding pocket, and the gene products of ~120 additional Bgh CSEPs very likely adopt a similar structure (Pennington et al., 2019). Structural similarity searches (IntFOLD Version 3.0) also suggested a ribonuclease-like fold for AVR_A7 and AVR_A13 (high and certain confidence at p=3.739E-3, and 6.174E-4, respectively), whereas no significant structural similarities were detected between CSEP0064 and AVR_A1, AVR_A9, AVR_A10, or AVR_A22 (low and medium confidence at p>0.01). Instead, we find that AVR_A9 may adopt a structural fold that is similar to an antimicrobial peptide called microplusin (p=6.014E-3). In addition, no obvious structural similarities were predicted between AVR_A1, AVR_A9, and AVR_A10/AVR_A22, suggesting that allelic MLA receptors are capable of detecting the presence of structurally unrelated Bgh effectors. This assumption is consistent with the recent finding that the wheat Mla ortholog Sr50 directly binds to the Basidiomycete stem rust (Pgt) avirulence effector AvrSr50 (Chen et al., 2017). This Basidiomycete effector most likely evolved independently from the Ascomycete Bgh effectors and lacks significant sequence and predicted structural similarity with the known AVR_a effectors. We speculate that MLA receptors might have an exceptional propensity to directly detect unrelated pathogen effectors and that this feature might have facilitated the functional diversification of the receptor in the host population.

In a whole leaf context, race-specific disease resistance specified by MLA receptors to Bgh is invariably linked to the activation of localized host cell death (Boyd et al., 1995). NLR-mediated cell death likely contributes to the termination of biotrophic fungal pathogenesis, including that of powdery mildews, because this class of pathogens feeds on living plant cells. A striking feature of the functional diversification at Mla is the enormous variation in microscopic and macroscopic resistance-associated Bgh infection phenotypes as shown with barley NILs carrying different Mla resistance specificities (Boyd et al., 1995). For instance, the onset of detectable host cell death can vary dramatically and can be both rapid and limited to the first attacked leaf epidermal cell, terminating early fungal pathogenesis, or can occur at later stages of fungal pathogenesis and involve numerous leaf mesophyll cells that subtend Bgh-infected epidermal cells (Boyd et al., 1995). Here we have employed co-transfection experiments of barley leaf protoplasts with Mla-AVR_a pairs and protoplast cell death as a proxy for receptor activation, excluding the possibility that additional Bgh-derived molecules associated with Pattern-triggered immunity influence the timing of immune receptor-mediated cell death in this system. Although based on overexpression data, the significant variation in cell death phenotypes reported here could partly reflect variable Bgh infection phenotypes on different MLA NILs (Supplementary file 1, Boyd et al., 1995). In turn, these differences of infection phenotypes are possibly due to variations in the steady-state levels of the MLA receptors during Bgh infection, timing of Bgh-mediated AVR_A secretion and/or AVR_A steady-state levels in planta, or MLA-AVR_A pair-dependent receptor binding affinities. Whilst etablishing the relevance of the the latter requires future biochemical characterization of MLA-AVR_A complexes, our work revealed a very strong binding of AVR_A13-V2 to MLA13 both in the split-LUC and yeast two-hybrid experiments (Figure 5), thereby uncoupling AVR_A binding to the receptor from receptor activation, that is immune receptor-mediated cell death activation. Future biochemical and genetic experiments will clarify whether the naturally occurring AVR_A13-V2 effector variant acts as a dominant negative ligand when co-expressed with the AVR_A13 avirulence effector.

RNA sequencing data have been deposited in GEO under accession code GSE110266 and improved Blumeria graminis f.sp. hordei isolate K1 assembly is deposited under the accession number PRJEB30373 at EBI-ENA. All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Isabel ML Saur

   Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5610-1260

2. Saskia Bauer

   Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4559-5063

3. Barbara Kracher

   Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Xunli Lu

   Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Present address

   Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing, China

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

5. Lamprinos Franzeskakis

   Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Aachen, Germany

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

6. Marion C Müller

   Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland

   Contribution

   Formal analysis, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5594-2319

7. Björn Sabelleck

   Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Aachen, Germany

   Contribution

   Methodology

   Competing interests

   No competing interests declared

8. Florian Kümmel

   Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Aachen, Germany

   Contribution

   Methodology

   Competing interests

   No competing interests declared

9. Ralph Panstruga

   Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Aachen, Germany

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3756-8957

10. Takaki Maekawa

    Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Contribution

    Formal analysis, Writing—original draft, Writing—review and editing

    Competing interests

    No competing interests declared

11. Paul Schulze-Lefert

    1. Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany
    2. Cluster of Excellence on Plant Sciences, Düsseldorf, Germany

    Contribution

    Conceptualization, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    schlef@mpipz.mpg.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8978-1717

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