| The cucumber(Cucumis sativus L.), belonging to the genus Cucumis of the family Cucurbitaceae, is grown in most temperate regions and represents the third most important vegetable crop in the world. Downy mildew, caused by Pseudoperonospora cubensis (Berk, and Curk.) Rostov., is a devastating foliar disease of cucumber epidemic particularly in humid conditions. Although fungicide can provide some level of the disease control, significant loss due to downy mildew has been addressed by both researchers and growers. The host resistance is therefore the most efficient and economic way to control the disease which largely depends on the understanding of inheritance of the resistance and the defense mechanism of the host. However, little information is available in cucumber above issues and the defense system of cucumber to downy mildew remains uncertain. To elucidate the genes associated with cucumber resistance against P. cubensis and provide a clue to look into the host defense mechanism, forty-two cultivars and inbred lines were employed in present research to select the resistant and susceptible materials, which were further used to construct the suppression subtractive hybridization (SSH) library. Through the reverse Northern dot-blot hybridization, sequencing and bioinformatics analysis, differentially expressed sequence tags (ESTs) were identified and classified. Moreover, the interaction between cucumber and P. cubensis was studied by monitoring the disease development and corresponding changes in cytochemistry and transcription of selected genes. The main results are as follows:1. Forty-two cucumber cultivars and inbred lines were chosen for the screening of downy mildew resistance. The assessment was conducted with seedlings by artificial inoculation in a greenhouse and with field-grown plants in two seasons by the natural disease epidemics. The results indicated that the disease index (DI) varied from 1.5 to 8.1 demonstrating a diverse genetic background of the cultivars and inbred lines tested. The cultivars and inbred lines, such as IL57, IL102, Jinchun 4, Nongcheng 4, Zhongnong 8 and Yuanfeng 6 were highly resistant to downy mildew according to the DI (DI< 2.5), among which inbred line IL57 was most resistant as it had the lowest DI. The susceptible varieties and inbred lines were IL101, IL112, Jinyou 3, Jinyou 30, New Ero and Changchun Mici as their IDs were above 6.0, in which inbred line IL112 was considered the most susceptible because of its ID was the highest among the materials tested. The IDs assigned to each cultivar or inbred line were quiet consistent among the three assessments and a highly significant correlation existed between the IDs of the seedlings and field-grown plants and between the spring and autumn field experiments. Therefore, inbred lines IL57 and IL112 were hence chosen in the subsequent experiments.2. Using mixed cDNAs prepared from 1157 seedlings induced with the pathogen 32,48 and 56 hours post inoculation (hpi), a forward subtractive cDNA library (FSL) and a reverse subtractive cDNA library (RSL) were constructed by the SSH. Based on the reverse Northern dot-blot screening, bioinformatics analysis to search by BLASTr and BLASTx and merging the repeated ones,60 and 26 singleton ESTs were isolated from the FSL and RSL, respectively. The ESTs with significant protein homology from the FSL were sorted into 12 functional categories, including protein synthesis, cellular transportation, primary metabolism, transcription, cell defense, energy metabolism, protein fate, proteins with binding functions, signal transduction, secondary metabolism, cell cycle and DNA processing, in addition to 14% of the ESTs with unknown function. The results revealed that cucumber genes associated with downy mildew are involved in possibally the whole process of the plant defense including signal transduction, cell defense, transcription, energy metabolism and cell rescue, etc.Besides the function-unknown genes, the singleton ESTs from the RSL were classified into 10 functional categories, such as energy metabolism, secondary metabolism, cell defense and rescue, proteins with binding functions, primary metabolism, protein synthesis, protein fate, cellular transportation and signal transduction. The down-regulated genes from the RSL would provide more information to decipher the defense mechanism.Among the differentially expressed genes, more (70%) were from the FSL and less (30%) from the RSL. More ESTs sorted into the energy and secondary metabolism were found in the RSL than in the FSL, indicating a depressing or damage of the corresponding metabolic processes under the pathogen stress.3. The downy mildew symptom in cucumber inbred lines IL57 and IL112 were investigated. Under any infected conditions, IL112 showed water-soaked lesions spread over the vines on the leaves which grew rapidly and resulted in a quick rotting of the leaves. In contrast, IL57 had less visible lesions than IL112 at early infection and chlorosis developed in the center of the infected spots which resulted in a localized tissue death at late stage of disease development with little further lesion spread within veins, showing a typical hypersensitive response (HR).The accumulation patterns of H2O2, callose and lignin were also histochemically determined in response to the infection of P. cubensis. H2O2 was firstly detected in attacked cells and its generation increased gradually in the cells surrounding the attacked sites of IL57. Accompanied with appearance of the HR, an early and abundant callose and lignin deposit were detected in resistant line IL57. The formation of H2O2, callose and lignin were also observed in IL112 but much later and less compared to IL57, indicating that the incompatible interaction between cucumber and P. cubensis is an important resistant type by which a rapid and abundant callose and lignin accumulation may be critical for inhibiting the mildew growth.To understand the mechanism of HR in cucumber to infection of P. cubensis, expression patterns of 34 selected genes in HR inbred line IL57 and susceptible inbred line IL112 were monitored by qRT-PCR. The results indicated distinct profiles between the two resistance types, most of the genes were early up-regulated (before 4 hpi) and abundantly expressed in IL57. An early and significant up-regulation of arginine decarboxylase gene, G protein gene and sterol 24-C-methyltransferase gene in polymine metabolic pathway implied that the initial phase of the oxidative burst and PCD could be triggered, at least partially if not completely, by these genes in the incompatible interaction between cucumber and P. cubensis. The suppression of ROS scavenging enzymes such as type-2 metallothionein and Mn-superoxide dismutase could contribute to the H2O2 accumulation. The galactional synthase, as a part in the scavenging mechanism, may be also necessary in HR to protect the plants from further damage of over loaded H2O2. Galactosidase and two other elongation factor 1-alpha genes may be involved in the cell wall modification as callose and lignin accumulated during HR.In summary, the present research first identified the hypersensitive response is a resistance type of cucumber against downy mildew. Our results provide cytological and molecular evidences that accumulation of H2O2 and deposition of callose are crucial mechanisms to confer a HR in cucumber. An early activation of the plant defense system is associated with HR and this coordination may be essential to ensure a high resistance level of the plant. |
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