Genome analyses of resistance-breaking isolates of the spinach downy mildew Peronospora effusa

Abstract

The biggest threat to spinach cultivation is the downy mildew Peronospora effusa (Pfs), a pathogen belonging to the oomycetes. Pfs is an obligate biotroph, meaning that it only grows on living host tissue. Although some fungicides are effective against this pathogen, the appearance of fungicide-resistance and reduction in use of agro-chemicals makes this form of protection less suitable. A more durable form of protection against this pathogen is by making use of genetic resistance mediated by resistance (R)-genes. To sustain an infection, the pathogen must, therefore, prevent or suppress immune responses of the host. Pfs, like other oomycetes, produces host-translocated effectors that can suppress defense responses or otherwise enhance susceptibility of the host. However, host-translocated effectors can also be recognized by R-proteins, and trigger plant immunity. This creates a strong selection pressure on the genome of the pathogen to adapt its recognized effector genes and thus avoid recognition. Alternatively, new effectors can evolve that can block the immune response. Overall, the use of R-genes on a wide scale has resulted in the rapid increase of Pfs isolates capable of breaking the employed resistances. To study the Pfs genes and effectors, the genome of race Pfs1 was sequenced and assembled. Due to the obligate biotrophic nature of this pathogen it cannot be cultured axenically, which led to the unintended sequencing of many other microbes present on the spinach leaves. To effectively filter sequences belonging to other microbes and remove them from the assembly, we used the CAT tool capable of determining the taxonomic origin of long error-corrected PacBio sequences and contig sequences resulting from a pre-assembly. This resulted in a clean Pfs1 genome assembly of 32.4 Mb. In addition Pfs1 mRNA was sequenced to aid gene prediction, resulting in 13,227 gene models. From the corresponding peptide sequences 99 RxLR(-like), and 14 Crinkler(-like) effector candidates were identified. Besides Pfs1, we also sequenced 16 denominated Pfs races and 8 field isolates. The identification of genomic variants between the Pfs isolates and the reference sequence (Pfs1) were used to determine the phylogenetic relationship between the isolates. The results indicate the presence of three distinct genetic groups, and one admixed group of Pfs isolates. The Pfs1 mitochondrial reference genome was assembled and used to identify two distinct mitochondrial haplotypes in the Pfs population. The genome and mitochondrial analyses showed that there is not a linear evolutionary relationship between the denominated Pfs races, and that sexual recombination has likely played an important role in the emergence of new Pfs races. We further found that effectors accumulate non-synonymous polymorphism at a higher rate compared to other genes in the genome. We also determined that two effector genes were absent in certain isolates understudy, of which the loss of one effector could be partially correlated to the breaking the RPF3-locus. Besides, amino acid substitutions in two effector proteins could be directly correlated to the breaking of RPF2, and substitutions in another effector were correlated to the breaking of RPF5.