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

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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.

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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

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Acknowledgments

This work was supported by the National High Technology Project of China (2007AA10Z177) and Key Projects in the National Science & Technology Pillar Program during Eleventh Five-Year Plan (2009BADB8B02). Authors are grateful to Dr. Tian Zhengdong and Dr. Song Botao from Huazhong Agricultural University for technical assistance.

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Authors and Affiliations

  1. Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Ministry of Education, Wuhan, 430070, Hubei, China

    Jian-Wu Li, Jun Liu & Cong-Hua Xie

  2. National Center for Vegetable Improvement (Central China), Wuhan, 430070, Hubei, China

    Jian-Wu Li, Jun Liu & Cong-Hua Xie

  3. Potato Engineering and Technology Research Center of Hubei Province, Wuhan, 430070, Hubei, China

    Jian-Wu Li, Jun Liu & Cong-Hua Xie

  4. Huazhong Agricultural University, Wuhan, 430070, Hubei, China

    Jian-Wu Li, Jun Liu & Cong-Hua Xie

  5. College of Horticultural Sciences, Henan Agricultural University, Zhengzhou, 450002, Henan, China

    Jian-Wu Li & He Zhang

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  1. Jian-Wu Li
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  2. Jun Liu
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  3. He Zhang
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  4. Cong-Hua Xie
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Correspondence to Cong-Hua Xie.

Additional information

Communicated by C. Quiros.

A contribution to the Special Issue: Plant Biotechnology in Support of the Millennium Development Goals.

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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)

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)

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)

Supplementary Table 1 (DOC 61 kb)

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Li, JW., Liu, J., Zhang, H. et al. Identification and transcriptional profiling of differentially expressed genes associated with resistance to Pseudoperonospora cubensis in cucumber. Plant Cell Rep 30, 345–357 (2011). https://doi.org/10.1007/s00299-010-0959-9

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  • DOI: https://doi.org/10.1007/s00299-010-0959-9

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