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Rapid transcriptional response of apple to fire blight disease revealed by cDNA suppression subtractive hybridization analysis

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Abstract

Fire blight, caused by the bacterium Erwinia amylovora, is a destructive disease of many tree and shrub species of the Rosaceae. Suppression subtractive cDNA hybridization (SSH) was used to identify genes that are differentially up- and down-regulated in apple (Malus x domestica) in response to challenge with E. amylovora. cDNA libraries were constructed from E. amylovora- and mock-challenged apple leaf tissue at various time intervals after challenge treatment, ranging from 0.25 to 72 h post-inoculation (hpi), and utilized in SSH. Gel electrophoresis of PCR-amplified SSH cDNAs indicated a greater quantity and size diversity in the down-regulated EST population at early times after challenge (1 and 2 hpi) compared to early up-regulated sequences and to sequences down-regulated at later (24 and 48 hpi) times after challenge. A total of 468 non-redundant Malus ESTs isolated by SSH in response to E. amylovora challenge were characterized by bioinformatic analysis. Many of ESTs identified following E. amylovora challenge of apple were similar to genes previously reported to respond to bacterial challenge in Arabidopsis thaliana. The results indicate that there was a substantial early (1 and 2 hpi) transcriptional response in apple to fire blight disease involving both the down- and up-regulation of host genes. Additionally, genes identified responding to fire blight challenge early (1 and 2 hpi) differed from those identified later (25, 48, and 72 hpi) in the infection process.

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Acknowledgments

We gratefully acknowledge Wilbur Hershberger (USDA, ARS, Kearneysville, WV, USA) for valuable assistance with conducting biological challenge experiments, isolating RNA from challenge tissues, cloning PCR-amplified SSH-cDNAs, and editing of SSH-EST sequences and for bioinformatic analyses; Dr. David Needleman (USDA, ARS, Wyndmoor, PA, USA) of the Eastern Regional Research Center’s Nucleic Acid Facility for sequencing the SSH-ESTs; Greg Richart (The Pennsylvania State University, York, USA) for assistance with SSH; Jing Ma (USDA, ARS, Kearneysville, USA) for assistance with semi-quantitative RT-PCR; John McGraw (USDA, ARS, Kearneysville, USA) for assistance with sequence editing and bioinformatics; and Dr. Zuping Yang (USDA-ARS, Kearneysville, USA) for assistance with cloning PCR-amplified SSH-cDNAs. The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005-35300-15462. Additional support was provided by NutriCore N.E.

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Authors and Affiliations

  1. Appalachian Fruit Research Station, USDA-ARS, 2217 Wiltshire Rd., Kearneysville, WV, 25430, USA

    John L. Norelli

  2. Biology Department, Pennsylvania State University, York, PA, USA

    Robert E. Farrell Jr.

  3. Appalachian Fruit Research Station, USDA-ARS, Kearneysville, WV, USA

    Carole L. Bassett, Donna A. Lalli & Michael E. Wisniewski

  4. Plant Genetic Resources Unit, USDA-ARS, Geneva, NY, USA

    Angela M. Baldo

  5. Department of Plant Pathology, NYSAES, Cornell University, Geneva, NY, USA

    Herb S. Aldwinckle

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  1. John L. Norelli
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  7. Michael E. Wisniewski
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Communicated by F. Gmitter

J. L. Norelli and R. E. Farrell, Jr. contributed equally to the design and execution of the experiments in this paper.

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Supplemental Table S1

List of PCR primers used to access expression of select ESTs by RT-PCR (DOC 41.5 kb)

Supplemental Table S2

Annotation of ESTs identified by SSH following E. amylovora challenge of ‘Gale Gala’ apple leaf tissue (XLS 123 kb)

Supplemental Table S3

Similaritya between SSH-ESTs identified following E. amylovora challenge of ‘Gale Gala’ apple leaf tissue (Malus SSH-EST) and 2,800 Arabidopsis genes differentially regulated in response to bacterial challenge (At-Bac gene) (XLS 105 kb)

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Norelli, J.L., Farrell, R.E., Bassett, C.L. et al. Rapid transcriptional response of apple to fire blight disease revealed by cDNA suppression subtractive hybridization analysis. Tree Genetics & Genomes 5, 27–40 (2009). https://doi.org/10.1007/s11295-008-0164-y

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