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 reassembled 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.

A Comprehensive Assessment of the Secretome Responsible for Host Adaptation of the Legume Root Pathogen Aphanomyces euteiches

“…Oomycetes produce CAZymes as a part of their arsenal for the supply of nutrition and to invade their preferential hosts. The GH repertoire may be linked to the oomycete lifestyle [19], with obligate biotrophic species having a reduced number and diversity of these proteins [97].…”

Pangenome graph analysis reveals extensive effector copy-number variation in spinach downy mildew

“…Figure 2B), which is a known challenge for biotrophic plant pathogens (Strong et al, 2014;Klein et al, 2020), yielding 29-100 contigs per isolate. These contigs were further scaffolded into chromosomes using the chromatin contact information from Hi-C data (S. Figure 4), and remaining gaps were subsequently closed by manually joining contig overlaps that were supported by long reads.…”

A Review of Chenopodium quinoa (Willd.) Diseases—An Updated Perspective

“…Progress in genome sequencing technologies can provide genome data to better understand how microbes live, evolve, and adapt. Indeed, the genome of three races of P. effusa (downy mildew of spinach) was recently sequenced, assembled, and annotated to gain insights into its gene repertoire and identify infection-related genes [ 9 ]. The genomes of microbial pathogens can vary greatly in size and composition; this also includes when closely related species are considered.…”

“…However, there is uncertainty when lower-level phylogenies (species level) are considered due to the fast-evolving traits and phenotypic plasticity of fungi [ 7 ]. As a result, DNA and molecular sequence-database comparisons techniques have been employed, along with various DNA fingerprinting and more advanced and complex methods such as whole-genome sequencing, for the identification of plant pathogens [ 8 , 9 ].…”