Genome reconstruction of the non-culturable spinach downy mildew Peronospora effusa by metagenome filtering.

Abstract

Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive many disease resistant cultivars have been bred and used. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences can, therefore, be considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed from the metagenome. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.4 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RxLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the predicted secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemi-biotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved in cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.