Twenty-five micrograms of proteins were allowed to wick up through the dipstick membrane

s outside the module. Using a Coefficient of Variation cutoff of 1.0, 4,195 genes from an initial total of 7,821 expressed genes were retained for downstream analyses. Using Weighted Gene Correlation Network Analysis , 3,146 genes were assigned to six different gene modules containing 107 to 1,312 genes; 1,049 8 mRNA-seq Analysis of Cucurbit Downy Mildew genes were not assigned to any module. Eigengenes were calculated for each module and displayed in a heat map revealing discrete gene expression patterns across different time points. As described above, the 1 dpi sample represents both an important “9357531 initial stage in the infection process, as well as a unique gene expression profile among the infection time points analyzed. This is additionally reflected in Module 1, which contains 146 genes that are highly expressed at 1 dpi, including genes involved in pathogenesis and transport. The genes in this module are also expressed at 3 dpi, indicating that there may be some similarities between processes involved in zoospore adhesion and encystment, and initiation “7644474 of haustoria formation. Genes expressed in Module 2 could represent those processes involved in the transition from early to late stages of infection and that are involved in the initial suppression of host defenses and establishment. This module, which contains 508 genes, expressed at 2, 3, and 4 dpi, represents gene expression occurring during initial penetration through the stomata into the host tissue, hyphal growth, and initiation of haustoria formation. It includes genes such as candidate RXLR-type effectors, glucanase inhibitors, CRNs, and a haustorium-specific membrane protein, similar to what has been observed to be up-regulated during P. infestans infection on potato. Our WGCNA analyses additionally identified genes that are co-expressed during the later stages of infection, specifically in Modules 4, 5, and 6. Module 6, in particular, represents genes most highly expressed at 8 dpi possibly indicative of those involved in a shift to the reproductive phase and sporulation. Transcription factors are reported to play a key role in the regulation of many biological processes, including roles in oomycete pathogenesis; within the predicted Ps. cubensis proteome, 27 transcription factor-related domains in 440 genes were identified. A total of 247 of these were expressed throughout the infection process. We also identified genes encoding transcription factor-related Pfam domains in all six co-expression modules. Two modules, 2 and 4, with genes co-expressed across different time contained the majority of the transcription factors. The transcription factorrelated genes within those modules could play important role in regulation of genes involved in pathogenesis. The bZIP and Myb, DNA-binding transcription factor, which play an important role in oomycete pathogenesis, were the most abundant transcription factor-related domains expressed during infection. Materials and Methods Ps. cubensis inoculation and sample collection Ps. cubensis MSU-1 was maintained on Cucumis sativus cultivar `Vlaspik’ as described previously. Four-week-old cucumber order A-83-01 plants were inoculated on the abaxial surface of the first fullyexpanded leaf with a 16105 sporangia/ml solution with 2030 10 ml droplets. Inoculated plants were maintained at 100% relative humidity in the dark for 24 hours and then transferred to growth chambers maintained at 22uC with a 12 h light/dark photoperiod. Samples were collected at 1,