Ultrathin sections were cut and post-stained with uranyl acetate and lead citrate

mily 12 endoglucanases have been shown to be up-regulated during early stages of infection. However, in H. arabidopsidis, which causes downy mildew of Arabidopsis thaliana, CWDE-encoding mRNAs are reduced. This could indicate an adaptation in downy mildew pathogens for evasion of recognition by their host, as break-down products from plant cell wall components can function as elicitors of defense responses. Recent advancements in sequencing technologies have led to an explosive growth in the analysis of in planta-expressed genes of biotrophic plant pathogens. In the current study, we present the first global gene expression analysis of the infection stages of cucumber by the obligate oomycete pathogen Ps. cubensis, the causal agent of cucurbit downy mildew. Through the analysis of a susceptible cucumber cultivar interaction, we describe the identification of genes with putative roles in infection, growth and pathogenicity. Using next-generation sequencing technology, we assessed gene expression in Ps. cubensis in sporangia and at six time points of infection. By combining visual assessment of symptoms with light microscopy to monitor infection stages as well as minimizing collection of non-inoculated tissues, we were able to capture expression of 7,821 Ps. cubensis genes ranging from 159 genes at 1 days post inoculation to 7,698 at 8 dpi. In total, this work represents a comprehensive examination of the key infection stages of Ps. cubensis growth and development. In total, the work described herein provides a foundation for further dissection of genes relevant to virulence in this obligate phytopathogen. Results and Discussion “1727148 Characterization and sampling of Ps. cubensis infection stages While Ps. cubensis is a major pathogen of cucumber and other cucurbits, limited MEK 162 resources describing the infection process and/ or virulence determinants of this obligate oomycete are available. In the current study, we sought to identify Ps. cubensis gene expression from both purified sporangia, as well as from a time course of infected cucumber tissues, representing a wide range of infection stages from 1 to 8 dpi. In total, our goal was to gain a broad perspective of in planta gene expression during infection of a susceptible cucumber host and to correlate this expression with the development of outwardly visible symptoms, as well as the development of microscopic pathogen infection structures. Like other phytopathogenic downy mildews and biotrophic fungi, Ps. cubensis is non-culturable, and proliferates and reproduces only on a susceptible cucurbit host. As with previously published reports on analyzing gene expression in biotrophic phytopathogens, optimization of sampling techniques is key to maximize pathogen tissue compared to host, particularly at early stages of infection . Plants were inoculated on the abaxial leaf surface with purified Ps. cubensis sporangia, and samples were collected using a cork borer, minimizing the amount of non-infected tissue in each sample. Initial symptoms of downy mildew infection “9357531 can be observed on the abaxial leaf surface at 13 dpi as water soaking at the site of inoculation, while no visual symptoms are apparent on the upper leaf surface. At 1 dpi, zoospores were encysted upon stomata on the lower leaf surface, and by 2 dpi, appressoria and initial penetration hyphae were visible beneath stomata. The yellow angular lesions typical of cucurbit downy mildew were apparent on the upper leaf surface by 4 dpi, and ove