The oomycete plant pathogen Phytophthora capsici infects several agriculturally important crop species. In cucumber (Cucumis sativus), P. capsici primarily causes fruit rot which is characterized by tissue collapse and dense mycelial growth. Previous studies in our lab have shown that some cucumber cultivars exhibit an age-related resistance (ARR) wherein young fruit are highly susceptible but develop resistance at approximately 12-16 days post pollination (dpp). Furthermore, the fruit peel has been shown to be important for conferring ARR and methanolic extracts from resistant peels had inhibitory effects on pathogen development. In the research herein, we sought to elucidate the mechanism controlling this ontogenic resistance in cucumber fruit by employing a diverse array of genetic, genomic, metabolomic and microscopic approaches. Using transcriptome analyses of peels from ARR expressing- and non-expressing cultivars, we identified unique upregulation of defense related factors in resistant-aged fruit peels, including resistance genes and transcription factors. An enrichment for genes involved in specialized metabolism in resistant fruit was also observed, and subsequently followed by an untargeted metabolomic analysis. We identified metabolites, specifically terpenoid glycosides, that may act as antimicrobial components in resistant-aged fruit peels. In a second study, we characterized the response to inoculation at resistant (8 dpp) and susceptible (16 dpp) ages via microscopic and transcriptomic analyses. Scanning electron microscopy of resistant peels showed evidence for infection failure as early as 4 hpi, including deflated or lysed spores and hyphae, that were not observed on susceptible fruit. Furthermore, transcriptome analysis of the first 48 hours post inoculation (hpi) revealed strong transcriptional defense responses at 4 hpi in both ages. At 24 and 48 hpi, susceptible 8 dpp fruit continued to mount defense along with strong downregulation of genes involved in photosynthesis and other biological processes. In contrast, resistant 16 dpp samples largely downregulated defense responses while upregulating photosynthesis. Weighted gene co-expression network analysis was used to further understand the transcriptional dynamics of infection during the first 24 hours. We identified early defense response modules which showed patterns of increased gene expression as early as 2 and 4 hpi, uniquely in resistant fruit. Several candidate genes involved in conferring this rapid response were identified. The early pathogen death and rapid defense response to infection in resistant-aged fruit indicate developmental changes that may include both pre-formed biochemical defenses and developmentally regulated capacity for pathogen recognition. To genetically map loci linked to ARR we employed a bulk segregant analysis approach. However, as no easy-to-use computational tools were available for these analyses, I developed a software package to use in our own experiments, but also as a tool for the plant breeding and genetics community. Using this tool, we analyzed bulks selected from two segregating populations derived from ARR expressing and non-expressing parents. We identified one locus on chromosome 3, linked to ARR, and using transcriptome data of the parental lines we identified a set of genes potentially associated with ARR. Together, these studies further our understanding of ARR as a biological phenomenon in general, as well as in the cucumber-P. capsici pathosystem specifically.
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