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Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa.

ABSTRACT: Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that 10 isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogen's reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding.

SUBMITTER: Skiadas P

PROVIDER: S-EPMC9304176 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.

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Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa.

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