Rapid turnover of effectors in grass powdery mildew (Blumeria graminis)
- Fabrizio Menardo1View ORCID ID profile,
- Coraline R. Praz1,
- Thomas Wicker†1Email author and
- Beat Keller†1Email author
© The Author(s). 2017
Received: 29 March 2017
Accepted: 2 October 2017
Published: 31 October 2017
Grass powdery mildew (Blumeria graminis, Ascomycota) is a major pathogen of cereal crops and has become a model organism for obligate biotrophic fungal pathogens of plants. The sequenced genomes of two formae speciales (ff.spp.), B.g. hordei and B.g. tritici (pathogens of barley and wheat), were found to be enriched in candidate effector genes (CEGs). Similar to other filamentous pathogens, CEGs in B. graminis are under positive selection. Additionally, effectors are more likely to have presence-absence polymorphisms than other genes among different strains.
Here we identified effectors in the genomes of three additional host-specific lineages of B. graminis (B.g. poae, B.g. avenae and B.g. infecting Lolium) which diverged between 24 and 5 million years ago (Mya). We found that most CEGs in B. graminis are clustered in families and that most families are present in both reference genomes (B.g. hordei and B.g. tritici) and in the genomes of all three newly annotated lineages. We identified conserved protein domains including a novel lipid binding domain. The phylogenetic analysis showed that frequent gene duplications and losses shaped the diversity of the effector repertoires of the different lineages through their evolutionary history. We observed several lineage-specific expansions where large clades of CEGs originated in only one lineage from a single gene through repeated gene duplications. When we applied a birth-death model we found that the turnover rate (the rate at which genes are deleted and duplicated) of CEG families is much higher than for non-CEG families. The analysis of genomic context revealed that the immediate surroundings of CEGs are enriched in transposable elements (TE) which could play a role in the duplication and deletion of CEGs.
The CEG repertoires of related pathogens diverged dramatically in short evolutionary times because of rapid turnover and of positive selection fixing non-synonymous mutations. While signatures of positive selection on effector sequences are the expected outcome of the evolutionary “arms race” between pathogen and plant immune system, it is more difficult to infer the mechanisms and evolutionary forces that maintained an extreme turnover rate in CEG families of B. graminis for several millions of years.
Basic statistics for the three new assembled Bg genomes
Number of contigs
B.g. on Lolium
Gene annotation in B. graminis
To compare the completeness of the annotations of the different genome assemblies the 458 core eukaryotic genes of Saccharomyces cerevisiae  were used as queries for blast searches against the protein databases of the five lineages of B. graminis that we annotated, and against the protein sequence databases of N. crassa and P. anserina. Genes with a blast hit with an e-value smaller than 10−7 were considered as present.
Identification of orthologous gene families and effectors in B. graminis
CEGs in B. graminis are normally identified through bioinformatic pipelines, originally they were defined as secreted proteins without homology outside of the Erysiphales , however the original set of candidate effectors has been expanded including genes with homology to the genes identified in Spanu et al. [10, 12, 14]. All these pipelines identify candidate effector genes, only extensive functional studies can validate these predictions. Here we used the pipeline of Praz et al.  to identify CEGs and CEG families as follow: the protein databases of Podospora anserina  and Neurospora crassa  were downloaded from www.blugen.org, http://fungi.ensembl.org/Podospora_anserina_s_mat_/Info/Index and www.broadinstitute.org/annotation/genome/neurospora/MultiDownloads.html (accessed 01/03/2014). After elimination of proteins with homology to transposable element proteins in the protein division of the TREP database (PTREP, blastp e-value cutoff ≤10–5, botinst.uzh.ch/en/research/genetics/thomasWicker/trep-db.html) (accessed 01/03/2014) we retained 9733 proteins for N. crassa and 10,604 for P. anserina.
Gene families are group of genes with sequence homology that evolved from a single gene through multiple duplications. To cluster proteins into families we performed all-against-all blast searches using protein sequences of N. crassa and P. anserina together with the protein sequences of all lineages of B. graminis obtained from Maker. Hits with e-value greater than 0.001 and alignment length shorter than 80% of the query length were excluded. Protein families were generated with the Markov cluster algorithm implemented in the software mcl 14–137 . We tried different values of the inflation parameter (1.4, 2, 4 and 6). We then chose to use 1.4 for further analysis because it yielded a more conservative (low) number of predicted effectors, thus limiting false positives. We identified CEGs as members of families unique to B. graminis (no members in N. crassa or P. anserina) and with at least one gene with a predicted signal peptide (SignalP 4.0) . We then manually inspected the list of families and their alignments and excluded four predicted effector families (55, 47, 8 and 8 genes) from subsequent analysis, because less than 15% of the genes (only one or two genes) harbored a predicted signal peptide.
Alignments and phylogeny
All protein alignments were performed with muscle 3.8.31 , maximum likelihood phylogenetic trees were inferred with RAxML 8.2.9  using a protein GTR model. Heterogeneity of the substitution rates among different sites was modeled with a discretized gamma distribution [38, 39]. In RAxML gaps are treated as missing data. Bootstrap support was computed with 1000 replications. The bootstrap convergence test implemented in RAxML showed that this was a sufficient number of replicates . To check for the presence of known functional domains we used all effectors in a blast search against the conserved domain database CDD  http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (e-value cut-off =0.01). Phylogenetic trees were visualized with Figtree 1.4.2.
Analysis of gene family evolution
We analyzed the evolution of gene families with CAFEv3.0 . CAFEv3.0 models gene gains and losses along a phylogenetic tree with a random birth-death process. We used the following species tree (obtained from Menardo et al. ) with branch lengths in million years: (((B.g. avenae:5,B.g. on Lolium:5):9,(B.g. tritici:8,B.g. hordei:8):6):11,B.g. poae:24) (Fig. 1). Given the species tree and the sizes of gene families, CAFEv3.0 can optimize the value of the birth-death rate λ to maximize the likelihood of the model given the observed data. All optimizations were run 10 times and the value that gave the highest likelihood was chosen. We estimated the turnover rate (λ) for all CEG families (49 families) and for all non-CEG families (434 families) with at least 10 genes among all lineages and then tested whether CEG families have the same turnover rate as non-CEG families. Turnover rates of single gene families were inferred with a single optimization process each. We then used CAFEv3.0 to simulate family sizes (using the most likely value of λ found for non-effector gene families) and tested which CEG families significantly deviate from the null expectation (that the variation of the family size fits with the distribution predicted by the null model of evolution, which was estimated from the non-CEG families).
The reconciliation analysis was performed on all gene families with at least 10 genes, we then compared the results of CEG families with non-CEG families. For this analysis we used ecceTERA  with standard parameters, the software computes the most parsimonious number of gene duplications, losses and transfers to reconciliate the gene tree with the species tree.
Analysis of genomic context of B.g. tritici genes
We used the high-quality B.g. tritici reference genome to compare the genomic context of CEGs and non-CEGs. For all 7825 predicted B.g. tritici genes, 5 kb upstream and downstream were extracted and TE annotation was performed by blastn seaches of the up- and downstream sequences against the B. graminis repeats contained in the TREP database. In total there are 92 known TE families in our in-house Blumeria graminis TE library. For this analysis we divided the 5 kb up and down stream of genes in 10 non-overlapping windows of 500 bp.
Effector families are conserved among all ff. spp. of B. graminis
Results of gene annotation and effector identification in B. graminis
Percentage of core eukaryotic genes annotated
Proportion of effectors in gene set
B.g. on Lolium
To identify candidate effector genes, we used mcl  to cluster all the predicted proteins in families together with the protein databases of the non-pathogenic ascomycetes Neurospora crassa and Podospora anserina. The fungi N. crassa and P. anserina have a saprophytic life style and therefore we assume they do not have any genes related to pathogenicity. Moreover, they are phylogenetically relatively close to Blumeria . We defined as effector families all families exclusive to B. graminis (no family members in N. crassa or P. anserina) and containing at least one gene coding for a protein with a predicted signal peptide (SignalP; ). We observed that not all CEGs were predicted to have a signal peptide (~ 38% of CEGs do not have a predicted signal peptide). This is partially due to incomplete coverage of some genes by the genome assemblies or by incomplete annotation. However, in most cases CEGs simply do not have a predicted signal peptide. It is possible that these genes are not functional, that they are not secreted and have a function within the fungal cell or that they are secreted through an alternative secretion system.
After manual curation, we identified 2798 predicted effector genes which clustered in 167 families. We found that CEG families (corresponding to ~ 83% of all effectors) contain genes from all the analyzed lineages of B. graminis. This finding implies that most CEG families were already present in the most recent common ancestor of all B. graminis lineages analyzed in this study, and therefore they originated before the divergence of B.g. poae from the other lineages (24 Ma). This also suggests that the role of most CEGs is conserved through all lineages of B. graminis which infect different plants, and is not specific for a single host. Additionally, we found that the genomes of B.g. avenae and of B. graminis growing on Lolium contain less CEGs than the other lineages (Table 2).
Conserved domains in effectors families
The 20 largest families of effectors in B. graminis
Bg tritici c
Bg hordei d
Bg avenae e
Bg on Lolium f
Bg poae g
We found several additional protein domains in B. graminis CEGs, they are involved in a multitude of different processes and it is often difficult to relate them to pathogenicity (Additional file 2: Table S1).
Evolution of effector families is shaped by gene duplications and losses
In a previous study , we reported on the evolutionary history of B. graminis and found that the earliest lineage to diverge was B.g. poa (about 24 Ma), followed about 14 Ma by a lineage that later gave rise to B.g. avenae and B. graminis infecting Lolium (about 5 Ma). Finally B.g. hordei and B.g. tritici diverged about 8 Ma (Fig. 1).
Rapid turnover of effectors in B. graminis
Genomic context of effector genes in B.g. tritici
Orthologous effector families in B. graminis
In this study we predicted effector genes in silico in the genome assemblies of five lineages of B. graminis with divergence times between 24 and 5 Ma. This represents a unique dataset, that compared to our previous study [10, 14, 26] has an expanded taxonomical and temporal breath, and it constitutes a vintage point to study the evolutionary processes that acted on CEGs in B. graminis over a long period of time. We found that more than 80% of the 2798 predicted effectors belong to families that include genes from all the analyzed lineages. Moreover, it was previously found that the great majority of B.g. hordei and B.g. tritici effectors do not have homologous genes in the genomes of pea (Erysiphe pisi) and Arabidopsis powdery mildew (Golovinomyces oronti) . These findings imply that most effector gene families of B. graminis originated in the period between the divergence of B. graminis from other mildews and the beginning of the differentiation of the different B. graminis lineages (22–83 Ma) . The finding that effectors are grouped in orthologous families suggests that their targets are also the product of homologous genes in the different grass and cereal host species. However, we observed that effectors of different lineages belonging to the same family are often very divergent at the sequence level due to positive selection and to the lineage-specific expansions and contractions of effector families. De Guillen et al.  found that effector genes without sequence homology of the ascomycete pathogen Magnaporthe oryzae have highly similar structures. It is therefore possible that also in B. graminis the functionality of effectors is determined mostly by the 3D structure of the protein rather than by the amino-acid sequence.
Conserved protein domains in effector families
Although most effectors do not have homology with known protein domains, we found that 209 of them have homology with a conserved protein domain. The most abundant was the RNAse domain which was already described in Pedersen et al. . Using template-based modeling of effectors, Pedersen et al.  also showed that it is possible to identify a structural homology to the RNAse domain for several additional effector families in B.g. hordei. Three of the characterized avirulence genes (AvrPm2 in B.g. tritici, Avr a13 and Avr a1 in B.g. hordei) and a putative suppressor of resistance (SvrPm3a1/f1 in B.g. tritici) belong to this class of RNAse-like effectors [28, 49–51]. However, Pedersen et al.  also found that the catalytic residues responsible for the RNAse activity are not conserved in B.g. hordei effectors. Additionally, we found two protein domains in B. graminis effectors, ML (6 genes) and CFEM (4 genes) domains, that have also been found to be enriched in the secretomes of two rust fungi (Melampsora larici-populina and Puccinia graminis f.sp. tritici) . These two rust pathogens are Basidiomycetes with an obligate biotrophic life style and they both form intracellular haustoria. The finding that these domains are present in the effector sets of the distantly related pathogen species rust and mildew suggests a convergent evolution of different organisms with a similar life style. While CFEM (cystein-rich fungal domain) has not been functionally characterized, the ML (MD2-related lipid-recognition) domain is present in many animal, plant and fungal proteins and is well studied . Proteins with this domain are usually shorter than 200 amino acids and involved in regulating lipid metabolism. In addition, they act as cofactors in recognition of pathogen-associated lipids and in the phospholipid transfer through membranes ). The well characterized protein from which the domain takes its name is MD-2. This protein interacts with Toll-like receptor 4 and it is necessary to recognize bacterial lipopeptides and lipopolysaccharides (LPS) in animals . Structural homology between some plant receptor-like kinase (RLK) and animal Toll receptors suggest similarities in the modes of action ; moreover, the receptor-like kinase Nt-Sd-RLK was shown to respond to bacterial LPS in Nicotiana tabacum . Among fungal membrane components, so far only ergosterol has been observed to be specifically recognized by plants , causing a typical elicitation response not caused by plant or animal sterols . Ergosterol is a perfect PAMP candidate because it is not present in plant membranes and can be recognized as non-self by the plant immune system . It is not known how plants recognize ergosterol, but one Arabidopsis RLK was shown to be responsive to others steroids (brassinosteroids) and to directly bind brassinolide . Therefore, we propose that the ML domain could be involved in the interactions occurring between the pathogen and host membranes, either blocking the recognition of fungal lipids by the plant cell receptors or interfering with the lipid signaling system of the plant.
Fast evolution of CEG families by gene duplication and loss
We observed that CEG families evolved following a birth-death model with an extremely high turnover rate compared to non-CEG families. This resulted in numerous gene deletions/disruptions and duplications that generated lineage-specific expansions and extinctions. To our knowledge no other study estimated the turnover rate in effector families. Pendleton et al.  found that in the rust fungus Cronartium quercuum f. sp. fusiforme (Basidiomycetes) CEG families underwent species-specific gene duplications. Moreover, Jiang et al.  and Goss et al.  reported repeated effector duplications in Phytophtora ramorum (Oomycetes), a pathogen with a broad host range.
The anomalous variation of CEG family sizes among lineages could be caused by natural selection or be the result of frequent structural modifications specific for CEG-containing regions. In the latter case, structural polymorphisms would be neutral and fixed by genetic drift. In B. graminis, effectors of the same family tend to weakly cluster in the same genomic region, but overall are found across the entire genome  (our own unpublished data). Based on this observation Pedersen et al.  proposed unequal crossing over as mechanism for repeated CEG duplications. It is possible that major structural rearrangements involved multiple members of the same family, causing fast expansions or reductions of some families (for example the CEG families 1, 2 and 7 with high turnover rate). Moreover, we found that the genomic context of CEGs has a high TE content, which could cause effectors to be duplicated and deleted more often than other genes in absence of selective pressure. The association of effectors with transposon-rich regions was observed also in other pathogens , and recently it was proposed that transposons have the major driving force in the adaptive evolution of the genome of fungal pathogen Verticillum dahliae .
It was shown before that CEGs in B. graminis and in other pathogens are exposed to two kinds of selective forces: the first is the necessity to maintain their virulence functions through time [10, 14]. The second is caused by the plant immune system: effectors can be recognized by immune receptors, which trigger an immune response that leads to the death of the pathogen (AvrPm3 a2/f2 and AvrPm2 in B.g. tritici, Avr a1 and Avr a13 in B.g. hordei) [28, 50, 51]. The effects of these two kinds of selection (escaping recognition and optimizing virulence) on the sequence of effectors are difficult to tear apart because in theory they both leave the typical signatures of positive selection . Only the molecular characterization of the interactions between effectors and their targets and between effectors and resistance proteins will clarify to what extent positive selection is due to either of the two mechanisms. Conversely, gene loss is a well-documented evolutionary mechanism to escape recognition from a resistance gene in many pathogens. In powdery mildew, two different mechanisms have been reported: complete gene deletion (AvrPm2 in B.g. tritici)  and insertion of unrelated DNA in the coding sequence (Avr a13 ) . It is tempting to speculate that the fast turnover rate that we observed in effector families of B. graminis is the direct consequence of the selective pressure of the plant immune system. Three of the four known Avrs in B. graminis belong to families with a large size variation and a high turnover rate (the exception is Avra1 in B.g. hordei which belongs to a family of only five genes that has a phylogenetic tree identical to the species tree). However, it is not clear whether selection or a neutral process (genetic drift) is the evolutionary force that drove the evolution of a high number of redundant effectors, which is a prerequisite to the possibility of losing them to escape recognition. Duplicated effectors could have given an immediate evolutionary advantage to the pathogen, impeding their molecular targets in the plant cell from evolving toward less susceptible variants: a plant target protein with a mutation that hampered the function of one effector would not have been fixed by positive selection because other slightly different effectors (resulting from a duplication and a subsequent mutation) were still functional. This is analogous to the strategy used in medicine and agriculture to prevent the evolution of drug-resistance in pathogens . Alternatively, flexible genomes and specifically flexible effector repertoires could provide an increased adaptive potential to the pathogen and be themselves a trait under selection.
We identified 2798 CEGs in the genome of five lineages of B. graminis with divergence times between 24 and 5 million years. We found that most of them cluster in families based on sequence similarity, and that most families are present in all lineages. Additionally, we found a high turnover rate in CEG families that resulted in large lineage-specific expansions and extinctions and contributed massively to the diversification of effector repertoires in lineages of B. graminis. Positive selection, previously described in Pedersen et al.  and Wicker et al. , and fast turnover act in parallel and are responsible for the fast evolution that we observe in effectors of B. graminis. These two processes caused a fast diversification of CEGs and are probably contributing to the adaptation of different lineages to different hosts. While positive selection fixes non-synonymous mutations, the fast turnover has two effects: it creates the effector repertoires on which positive selection can act (gene duplication), but at the same time destroys genes, contributing to increase the diversity of effector repertoires in pathogen populations.
Availability of data and materials
Genome assemblies, CDS databases and alignment of the ML domain are available as Additional files.
This work was supported by the University Priority Program “Evolution in action” of the University of Zurich and the grant 310,030–163,260 from the Swiss National Science Foundation.
BK designed the study and wrote the manuscript, TW and FM designed the study, performed the analysis and wrote the manuscript, CRP performed the analysis. All authors read and approved the final manuscript.
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