Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora

Abstract

Background: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.

Results: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.

Conclusions: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

Fig. 1

Genome assembly quality assessment in terms of length of the shortest scaffold in each N-class and the number of scaffolds. The quality of the genome assembly was assessed by first sorting all 3143 nuclear scaffolds length-wise from the largest to the smallest scaffold. Then N-classes were defined, where N represents the percentage of genome covered by considering the assembled genome size. The length given for each N-class represents the length of the smallest scaffold present in that particular N-class. The number of scaffolds represents the number of scaffolds present in the respective N-class. The sharp rise after N98 represents the unresolved small contigs, the majority of which are repeat elements

Fig. 2

Genome completeness and continuity assessments in terms of core housekeeping genes. Genome completeness in terms of core eukaryotic genes was assessed using the CEGMA pipeline. The CEGMA pipeline has categorized 458 core genes into 4 groups on the basis of their conservation, from the least conserved group 1 to the most conserved group 4. a Genome completeness in terms of complete mapping. b Genome completeness estimations in terms of partial mapping

Fig. 3

Phylogenetic relationship of deeply sequenced oomycetes. The phylogenetic analysis was done by considering the core orthologous genes predicted by the CEGMA pipeline. Multiple sequence alignments were performed using Mafft and phylogenetic relationships were inferred using the Maximum Likelihood algorithm as implemented in RAxML. Number on branches correspond to support values from 1000 bootstrap replicates

Fig. 4

Number of ortholog groups within oomycete genomes. The number of ortholog groups among the genomes of Hy. arabidopsidis, Ph. capsici, Ph. infestans, Ph. sojae, and Pl. halstedii. a Number of ortholog groups found within the five genomes considering all protein-coding genes. b Number of ortholog groups within the five genomes considering all PSEP-encoding genes. Numbers in brackets represent the total number of genes tested in the analyses. Asterisks denote 1:1 orthologs among the five genomes

Fig. 5

Heat maps illustrating gene density of the Pl. halstedii genome. Gene density as estimated by calculating the 5′ and 3′ flanking distances of (a) all protein encoding genes, (b) core genes (c) non-secreted protein encoding genes (d) secreted protein encoding genes, (e) candidate RxLR-like protein encoding genes, (f) CRN-like protein encoding genes. Grey shading highlights the area with both 5′ and 3′ distances below 3 kb

Fig. 6

Features of promoters. a A + T content of coding regions and 50-nt intervals within promoters from Pl. halstedii, Ph. infestans, and Hy. arabidopsidis. b Distribution of motifs in different Straminipila. Searches for the INR + FPR supra-motif, INR, FPR, and DPEP were performed in five oomycetes (Ph. infestans, Pl. halstedii, Hy. arabidopsidis, Py. ultimum, Sa. parasitica) and the diatom Thalassiosira pseudonana. Bars show the percentage of promoters within each species that contain the motifs within 200 nt of the start codon, corrected for false discovery. The figure on the left is a neighbor-joining tree based on ribosomal RNA and internal transcribed spacer (ITS) sequences. c Positional bias of INR + FPR supra-motif and CCAAT within Pl. halstedii promoters. The right of the panel compares the content of the two motifs in Ph. infestans and Pl. halstedii

Fig. 7

Features of RxLR-dEER-like effectors and frequency of the RxLR and RxLR-dEER-like proteins in the genome of Pl. halstedii: a Sequence features of the RxLR-dEER-like proteins were calculated from predicted putative RxLR-like proteins. Numbers in brackets represent the minimum and maximum values of distances and number in italics represents the corresponding mean value. Multiple sequence alignments were performed by using Mafft and sequence logos were generated using jalview. b Bar plot representing the number of RxLR-like and RxLR-dEER-like proteins in the predicted secretome of Pl. halstedii

Fig. 8

Orthologs of RxLR-dEER-like proteins within downy mildew pathogen genomes and Phytophthora spp. genomes: High confidence RxLR-dEER-like proteins from the secretome of downy mildew and Phytophthora spp. genomes were predicted and orthology analyses were performed with OrthoMCL to predict orthologs of RxLR-dEER-like proteins. Pha, Hpa, Pca, Pin, Pso, and Prm refer to Pl. halstedii, Hy. arabidopsidis, Ph. capsici, Ph. infestans, Ph. sojae, and Ph. ramorum, respectively. a Venn diagram showing the number of orthologs among the four Phytophthora spp. genomes. b Table summarising the number of orthologs shared by downy mildews and Phytophthora spp. genomes. c Sequence alignments of the three candidate orthologs of putative RxLR-dEER proteins among the six genomes. Multiple sequence alignments were performed using Mafft and alignment graphics were generated using Jalview. Cleavage sites predicted by SignalP are highlighted by red circles, RxLR/dEER-like motifs are highlighted by red boxes