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

Abstract

<jats:p><jats:italic>Peronospora effusa</jats:italic> (previously known as <jats:italic>P. farinosa f. sp. spinaciae</jats:italic>, and here referred to as <jats:italic>Pfs</jats:italic>) is an obligate biotrophic oomycete that causes downy mildew on spinach (<jats:italic>Spinacia oleracea</jats:italic>). To combat this destructive disease resistant cultivars are continually bred. However, new <jats:italic>Pfs</jats:italic> races rapidly break the employed resistance genes. To get insight into the gene repertoire of <jats:italic>Pfs</jats:italic> and identify infection-related genes, the genome of the first reference race, <jats:italic>Pfs1</jats:italic>, 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 are, therefore, considered a metagenome. To filter and obtain <jats:italic>Pfs</jats:italic> 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. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.40 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 secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemibiotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of <jats:italic>Pfs1</jats:italic> will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.</jats:p>

Ancestral chromosomes for the Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa

“…Genome-wide, 3.1% of the genes are annotated as effectors; therefore, effectors were enriched in the gene-sparse regions of the assembly. The upper limit of this analysis was better than a previously reported similar analysis of P. effusa Pfs1 (Klein et al, 2020), likely because the Pfs1 assembly was more fragmented than the T2T UA202013 assembly. The two-speed genome hypothesis proposes that effectors are embedded in gene-sparse regions of the genome (Dong et al, 2015).…”

“…To date, there are 19 named races of P. effusa; however, isolates with variation in virulence phenotype are continuously identified and the biology behind the emergence of new races is not clearly understood (Plantum, 2021, April 15). Previous genomic investigations have yielded fragmented and repeat-sparse genome assemblies (Feng et al, 2018a;Fletcher et al, 2018;Klein et al, 2020). The number of chromosomes the genome of P. effusa contained, the number and genomic distribution of genes encoding virulence factors, and how much of the genome is conserved or variable between isolates remained unknown.…”

Genome-wide simple sequence repeats (SSR) markers discovered from whole-genome sequence comparisons of multiple spinach accessions

“…These new SSR markers may also help develop near isogenic lines (NIL) to track the resistance gene introgressed region of the recurrent susceptible lines. Furthermore, the genome of some races of spinach downy mildew pathogen ( Peronospora effusa race 1, 12, 13, 14) has been sequenced 67 – 69 . These sequences could be searched to identify the set of SSRs varying among the races, identify a genome-wide fingerprint and diagnostic sets of SSR loci, and identify SSRs involved on and/or associated with virulence-pathogenic loci.…”

The Effector Repertoire of the Hop Downy Mildew Pathogen Pseudoperonospora humuli

“…The finding of bacterial sequences in the P . cubensis proteome affirms the need for specialized laboratory protocols that account for the phyllosphere microbiome and stringent bioinformatics filtering in initial steps when dealing with the genomic data of biotrophic pathogens ( Klein et al, 2019 ). With the range of sequencing technologies now available, resequencing the P .…”

“…The presence of contaminants is not surprising in next generation sequencing data from biotrophic pathogens like P. cubensis and P. humuli (Laurence et al, 2014;Rahman et al, 2019). The finding of bacterial sequences in the P. cubensis proteome affirms the need for specialized laboratory protocols that account for the phyllosphere microbiome and stringent bioinformatics filtering in initial steps when dealing with the genomic data of biotrophic pathogens (Klein et al, 2019). With the range of sequencing technologies now available, resequencing the P. cubensis genome with clean material and the use of in silico methods to remove contaminants might result in more accurate RXLR predictions.…”