Plant Cell Reports

, Volume 30, Issue 3, pp 345–357

Identification and transcriptional profiling of differentially expressed genes associated with resistance to Pseudoperonospora cubensis in cucumber

Original Paper

DOI: 10.1007/s00299-010-0959-9

Cite this article as:
Li, JW., Liu, J., Zhang, H. et al. Plant Cell Rep (2011) 30: 345. doi:10.1007/s00299-010-0959-9

Abstract

To identify genes induced during Pseudoperonospora cubensis (Berk. and Curk.) Rostov. infection in cucumber (Cucumis sativus L.), the suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from cucumber seedlings inoculated with the pathogen as a tester and cDNA from uninfected cucumber seedlings as a driver. A forward subtractive cDNA library (FSL) and a reverse subtractive cDNA library (RSL) were constructed, from which 1,416 and 1,128 recombinant clones were isolated, respectively. Differential screening of the preferentially expressed recombinant clones identified 58 unique expressed sequence tags (ESTs) from FSL and 29 from RSL. The ESTs with significant protein homology were sorted into 13 functional categories involved in nearly the whole process of plant defense such as signal transduction and cell defense, transcription, cell cycle and DNA processing, protein synthesis, protein fate, proteins with binding functions, transport, metabolism and energy. The expressions of twenty-five ESTs by real-time quantitative RT–PCR confirmed that differential gene regulation occurred during P. cubensis infection and inferred that higher and earlier expression of transcription factors and signal transduction associated genes together with ubiquitin/proteasome and polyamine biosynthesis pathways may contribute to the defense response of cucumber to P. cubensis infection. The transcription profiling of selected down-regulated genes revealed that suppression of the genes in reactive oxygen species scavenging system and photosynthesis pathway may inhibit disease development in the host tissue.

Keywords

Cucumis sativus L. Downy mildew Suppression subtractive hybridization Resistance-related genes 

Abbreviations

ESTs

Expressed sequence tags

FSL

Forward subtractive library

qRT–PCR

Real-time quantitative RT–PCR

ROS

Reactive oxygen species

RT–PCR

Reverse transcription-polymerase chain reaction

RSL

Reverse subtractive library

SSH

Suppression subtractive hybridization

Supplementary material

299_2010_959_MOESM1_ESM.tif (282 kb)
Supplementary Fig. 1 A representative sample of reverse Northern dot blot screening of clones from the forward subtractive library. A represents the result of membrane hybridization with the tester probe. B represents the result of membrane hybridization with the driver probe. A 18s ribosomal cDNA was used as the positive control (a 1, a 2, a 3, a 10, a 11, a 12, f 4, f 5, f 6, f 7, f 8, f 9, k 1, k 2, k 3, k 10, k 11, k 12), and ddH2O as the negative control (a 4, a 5, a 6, a 7, a 8, a 9, f 1, f 2, f 3, f 10, f 11, f 12, k 4, k 5, k 6, k 7, k 8, k 9). The circles indicate the corresponding clones enriched after inoculation (TIFF 282 kb)
299_2010_959_MOESM2_ESM.doc (60 kb)
Supplementary Fig. 2 Functional categories of (A) up- and (B) down-regulated genes in resistant inbred line IL57 after infection with Pseudoperonospora cubensis (DOC 59 kb)
299_2010_959_MOESM3_ESM.doc (93 kb)
Supplementary Fig. 3 Photorespiratory pathway and C3 carbon cycle induced in resistant inbred line IL57 under Pseudoperonospora cubensis infection. The mRNA expression level were validated by real-time quantitative RT-PCR for gene 6P03 encoding Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), 7F08 encoding aminotransferase and 7J03 encoding NAD+- hydroxypyruvate reductase. GLK, glycerate kinase; GLO, glycolate oxidase; 2-PHP, 2-phosphoglycolate phosphatase; SHM, serine hydroxymethyltransferase (DOC 93 kb)
299_2010_959_MOESM4_ESM.doc (62 kb)
Supplementary Table 1 (DOC 61 kb)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Jian-Wu Li
    • 1
    • 2
    • 3
    • 4
    • 5
  • Jun Liu
    • 1
    • 2
    • 3
    • 4
  • He Zhang
    • 5
  • Cong-Hua Xie
    • 1
    • 2
    • 3
    • 4
  1. 1.Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural UniversityMinistry of EducationWuhanChina
  2. 2.National Center for Vegetable Improvement (Central China)WuhanChina
  3. 3.Potato Engineering and Technology Research Center of Hubei ProvinceWuhanChina
  4. 4.Huazhong Agricultural UniversityWuhanChina
  5. 5.College of Horticultural Sciences, Henan Agricultural UniversityZhengzhouChina

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