Project description:Using next generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen
Project description:Cucumber (Cucumis sativus L.) is an economically important vegetable crop distributed in over 80 countries. Downy mildew (DM) caused by the obligate oomycete Pseudoperonospora cubensis is especially destructive in cucumber production. So far, few studies on the changes in proteomes during the P. cubensis infection have been performed. Using a newly developed TMT-LC-MS/MS analysis, the proteomes of DM-resistant variety ‘ZJ’ and DM-susceptible variety ‘SDG’ under the P. cubensis infection were investigated. In total, 6400 proteins were identified, 5629 of which were quantified. The differential accumulated proteins (DAPs) exhibited various biological functions and diverse subcellular localizations. KEGG enrichment analysis showed that various metabolic pathways were significantly altered under the P. cubensis infection, such as terpenoid backbone biosynthesis, and selenocompound metabolism in ZJ, and starch and sucrose metabolism in SDG. Most of the enzymes associated with terpenoid backbone synthesis were significantly accumulated in ZJ rather than in SDG, suggesting that pathogen-induced terpenoids accumulation might play an important role in the resistance against P. cubensis infection. Furthermore, a number of pathogenesis-related proteins and heat shock proteins were identified as DAPs, suggesting that DM resistance was controlled by a complex network. Our data allowed us to identify and screen more potential proteins related to the DM resistance.
Project description:We used Bio-Rad laboratories, lnc. PCR assay panel to analyze expression levels of MAPK genes under various stress conditions and plant hormone treatments. Cucumber plants of the ‘Jinyan No.4’ cultivar were reared in growth chambers at 28 ± 1 °C with a photoperiod of 16 h light/8 h dark and light intensity of 400 μmol m−2 s−1. Three-week-old seedlings were used for all abiotic and biotic treatments. For heat or cold treatment, the seedlings were subjected to 35 ± 1 °C or 4 ± 1 °C conditions, respectively. The samples for RNA extraction were collected at 0, 1, 2, 4, and 8 h after treatment. For dehydration treatment, the leaves of plants were sampled at 0, 2, 4, and 6 d after watering stopped. For disease treatment, Pseudoperonospora cubensis (P.cubensis) was used to infect the seedlings, and leaves were collected at 0, 1, 2, and 3 d after infection. For hormone treatments, the seedling leaves were sprayed with 100 mM methyl jasmonate (meJA) or 100 mM abscisic acid (ABA) and then sampled at 0, 1, 2, 4, and 8 h intervals. The experiment was designed to examine the transcription patterns of 58 MAPK cascade genes in cucumber in response to three different abiotic stresses (cold, heat, and drought), one biotic stress (Pseudoperonospora cubensis) and two (MeJA and ABA) plant hormone treatments.
