Pseudoperonospora cubensis [(Berkeley & M. A. Curtis) Rostovzev] is the causal agent of cucurbit downy mildew, a foliar disease responsible for devastating losses worldwide of cucumber, cantaloupe, pumpkin, watermelon, and squash. In the United States, cucurbit downy mildew is the major threat to cucumber (Cucumis sativus) production and has been a significant limiting factor since the 2004 and 2005 growing seasons. Prior to 2004, host resistance had been an effective means of controlling the disease and, as such, limited research had been conducted to study the biology, genetics, or virulence of Ps. cubensis. The first step to expand the knowledge base of Ps. cubensis was the generation of a 64.4 Mbp genomic assembly of the MSU-1 isolate and prediction of 23,519 loci and 23,522 gene models. Similar to other oomycete plant pathogens, Ps. cubensis utilizes RXLR and RXLR-like effector proteins, which can function as either virulence or avirulence determinants during the course of host infection. Using in silico analyses, 271 candidate effector proteins were identified with variable RXLR motifs, including 20 different amino acids at position R1. Of these, only 109 (41%) had a putative ortholog in Phytophthora infestans and evolutionary rate analysis of these orthologs shows that they are evolving at a significantly faster rate than most other genes. One Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1) was characterized in detail. PscRXLR1 was shown to be up-regulated during the early stages of host infection, and elicits a cell death response in Nicotiana benthamiana. PscRXLR1 was also demonstrated to be a product of alternative splicing, marking this as the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene into a functional effector protein. We present the first large-scale global gene expression analysis of Ps. cubensis infection of a susceptible C. sativus cultivar, `Vlaspik', and identification of both pathogen and host genes involved in infection and the defense response, respectively. Using mRNA-Seq, we captured differential expression of 2383 Ps. cubensis genes in sporangia and at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Co-expression analyses identified distinct modules of Ps. cubensis genes that were representative of early, intermediate, and late infection stages. Additionally, the expression of 15,286 cucumber genes was detected, of which 14,476 were expressed throughout the infection process from 1 dpi to 8 dpi. The rapid induction of key defense related genes, including catalases, chitinases, lipoxygenases, peroxidases, and protease inhibitors was detected within 1 dpi, suggesting recognition by C. sativus of the initial stages of Ps. cubensis infection. Co-expression network analyses revealed transcriptional networks with distinct patterns of expression including down-regulation at 2 dpi of known defense responsive genes suggesting coordinated suppression of host responses by the pathogen. In total, the work described herein presents an in-depth analysis of the interplay between host susceptibility and pathogen virulence in an agriculturally important pathosystem.
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