Structure and evolution of barley powdery mildew effector candidates

Abstract

Background: Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.

Results: Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen's annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100-150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300-400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.

Conclusions: We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.

Figure 1

Summary of bioinformatics and expression analysis of CSEPs. Identification of CSEPs and family assignment. Workflow used to find and annotate CSEPs in the genome of B. graminis f.sp. hordei. After three iterative rounds of BLAST and annotation, genes were clustered into families as described in Methods. Protein analyses. The proteins predicted to be translated from the CSEP ORFs were analyzed to infer their 3D structure and presence of conserved motifs. These were then used to investigate evidence of different selection pressures during the course of evolution of the CSEP families. Expression analysis. Evidence to support the existence of CSEP genes was obtained from ESTs in the public databases, from RNAseq surveys and from the analysis of the proteomes of B. graminis and B. graminis-infected barley tissues by mass-spectrometry.

Figure 2

CIRCOS plot of the CSEP superfamily with expression and proteome data. From the perimeter to the centre: The outer ring identifies the CSEPs. The rectangles in the circle immediately below the identifiers are colour-coded: CSEPs of the same families have the same colour. The small circles below the family identifiers indicate the proteins identified by mass spectrometry in infected epidermis only (green) or in both infected epidermis and epiphytic hyphae (yellow). The first and second data histogram circles shows the expression values of the haustorial samples (blue) and of the epiphytic samples (red) of each CSEP gene on a log₂ scale. The third data histogram (black) represents the ratio of the expression values in the two stages plotted on a log₂ scale. The fourth data circle indicate the statistical significance of the ratios (red, significant/black non-significant). At the centre is a dendrogram based on the neighbour-joining dendrogram of all CSEP paralogs.

Figure 3

Protein structure and positive selection in CSEP family 12.A: Amino acid alignment of the seven members obtained with CLC main workbench (see Methods). B: Evidence for selection on the paralog members of family 12 was estimated using the Selecton server ( [49,50]; http://selecton.tau.ac.il/). Codon sites under positive diversifying (red) or purifying (purple and yellow) selection and conserved cysteines (yellow) are indicated by coloured circles. C: Cladogram with Ka/Ks-values indicated for the individual branches calculated using the on-line server at http://services.cbu.uib.no/tools/kaks. Branches in red indicate a significant positive selection. D: 3D protein models of two family 12 members are shown and the amino acids under positive diversifying selection are highlighted in red.

Figure 4

The relationship between the length of CSEPs, the degree of positive selection and the ratio of expression in haustoria compared to expression in epiphytic hyphae. Ratio of the non-synonymous to synonymous substitutions (Ka/Ks) within CSEP families is plotted against the length of the proteins. The values of the parameters for the axes were calculated as family averages; the family numbers are indicated in the circles. The ratios of CSEP expression in haustoria and epiphytic hyphae are as indicated in the colour bar. The diameter of the circles indicates the relative size of the families.

Figure 5

Multiple sequence alignment and 3D models of ribonucleases and CSEPs. A: Multiple sequence alignment of ribonuclease T1 from Aspergillus oryzae, a ribonuclease consensus sequence and selected CSEP family consensus sequences. The ribonuclease consensus was derived by aligning ribonucleases from Aspergillus phoenicis (P00653, Penicillium brevicompactum (P07446), Grosmannia clavigera (EFX05096), Phaeosphaeria nodorum (XP_001800520) and Mycosphaerella graminicola (EGP89360). The alignments were manually edited based on MultAlin-alignments (http://multalin.toulouse.inra.fr/multalin/multalin.html). The CSEP families included are primarily those showing most ribonucleases identified by InterProScan or by the structural annotation. The secondary structures (α-helix, β-sheets and loops) of ribonuclease T1 from Aspergillus shown on top are according to Pace et al. [54]. Catalytic active site residues in ribonucleases are indicated in red. Intron position is indicated by a red vertical dashed line; there is one exception, one member of family 56 does not have this intron. Amino acid numberings are the ranges for each family. Upper case letters indicate highly conserved positions, while lower case letters indicate that the positions are present in some of the family members only. Omega (Ω) is used for aromatic amino acids (F, Y and W), and psi (Ψ) is used for V, L and I. Letters in bold indicate that the positions are under purifying selection. Dots indicate non-conserved positions and dashes are gaps. B: 3D models of ribonuclease T1 and three CSEPs and their superposition. Arrows indicate the predicted disulphide bonds between the N- and C-terminal cysteines.

Figure 6

Genome clustering of eight CSEP paralogs from family 7 on two sequence scaffolds. A: Correlations between the phylogenetic relationships based on nucleotide sequences of CSEP paralogs from family 7 and their locations on the genomic sequence scaffolds 005496 (red) and 005502 (blue) indicated with dotted lines. Only the relevant parts of the sequence scaffolds (scale bar) are shown. The colour code of the CSEPs refers to the genomic organization shown in panel B. B: Schematic illustration of the genomic organization (encompassing about 5 kb) of the eight CSEP paralogs with the retro-transposable elements Egh24 and Eg-R1[15,16]. The percentages of nucleotide sequence identity in pairwise comparisons are indicated and abrupt changes in sequence similarity are indicated with vertical dashed lines in red. The colour code of the CSEPs refers to the phylogenetic tree shown in panel A. Scale bars represent lenghts of DNA in base pairs.