Cucumber mildew resistance: identification of cucumber genes involved in susceptibility and resistance to powdery and downy mildew

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Two of the most limiting diseases in cucumber production are downy mildew (DM), caused by the oomycete Pseudoperonospora cubensis, and powdery mildew (PM), caused by the fungi Podosphaera xanthii and Golovinomyces orontii. Whereas the pathogens causing DM and PM are not related, they share a similar obligate biotrophic lifestyle. In my PhD thesis we aimed at identifying genes involved in susceptibility and resistance to these diseases, in order to better understand the interactions between cucumber and mildew pathogens, and contribute to breeding disease resistant cucumbers. One focus point in my research was the concept of susceptibility (S) genes. Loss-of-function of S-genes lead to resistance, which is more durable than classical race-specific resistance (R) genes. A second pillar of my PhD research was to identify causal genes for QTL contributing to disease resistance, which are frequently used in cucumber breeding. Regarding PM, the most important source of resistance is “hypocotyl resistance”, which causes partial resistance characterized by PM-free hypocotyls and stems, and can lead to full resistance when combined with other sources of resistance. One of the most famous examples of an S gene is MLO, loss-of-function alleles of which provide durable PM resistance in barley already for several decades. After the cloning of the causal gene it was found that MLO genes occur in all sequenced plant genomes to date, and that in several plant species MLO homologues in phylogenetic clades IV and V were found to be S genes for PM. In the first two chapters of my thesis I investigated MLO genes in cucumber, leading to the identification and functional characterization of a loss-of-function allele of the clade V CsaMLO8 gene as causal for hypocotyl resistance in cucumber (Chapter 2). Furthermore, we functionally characterized the other clade V MLO genes in cucumber, CsaMLO1 and CsaMLO11, and found that both are functional S-genes too, although they are rather weakly expressed in leaves, presumably leading to a minor role regarding PM susceptibility (Chapter 3). In the second half of my thesis the focus is on identification of genes involved in DM resistance. Currently, the most often used source of DM resistance in cucumber is the Indian semi-wild accession PI 197088, the resistance in which inherits as a polygenic trait. We introgressed one major QTL from this resistant accession in a uniform susceptible background, in order to fine-map it. We found that this QTL consists of several subQTL, each explaining a different aspect of the resistance conferred by the full QTL. Through a combination of transcriptomics and whole genome sequencing, we identified likely candidate genes for two of these subQTL. For subQTL DM4.1.2, which had a dominant effect on sporulation, we found a novel RLK gene (CsLRK10L2) which was strongly upregulated by the pathogen. This gene has homology to LRK10 genes, which were originally found as candidate genes for leaf rust resistance in wheat. This LRK10-like gene had a 551 bp deletion in DM susceptible genotypes compared to resistant genotypes. The cucumber reference genome also shows this deletion. The presence of a predicted oligogalacturonan-binding domain in the CsLRK10L2 protein suggested that this receptor might be involved in sensing cell-wall damage, triggering a defence response (Chapter 4). For subQTL DM4.1.3, which had an effect on both chlorosis and sporulation, we identified a mutation in the Amino Acid Permease 2A (CsAAP2A) gene, encoding a transporter for amino acids. Homologs of this gene were previously found to be S-genes for several obligate parasitic nematode species, as they heavily rely on their host to provide them with amino acid nutrients. We found that cucumber plants with the loss-of-function allele of CsAAP2A contained lower levels of amino acids after inoculation compared to WT plants, indicating that the mutation might decrease amino acid transport to infected leaves. As such, this gene is a novel S-gene for oomycetes (Chapter 5). Combined, the results described in this thesis increase our knowledge about the interactions between cucumber and two of its most notorious pathogens, and will facilitate cucumber resistance breeding.