Functional Genomic Response of Apple to Fire Blight


John (Jay) Norelli (USDA-ARS), Herb Aldwinkle (Cornell), Carole Bassett (USDA-ARS-AFRS), James Farrell, Jr. (Penn State).
Phone: 304-725-3451
Fax: 304-728-2340
Email: jnorelli@afrs.ars.usda.gov


Accomplishments:
  • ESTs associated with apple’s response to fire blight have been identified by SSH, cDNAAFLP and bioinformatics.
    • SSH identified a rapid transcriptional response of apple to fire blight challenge 1 hour post inoculation (hpi). 33% of all ESTs identified in apple following fire blightchallenge (1 to 72 hpi) had high similarity (e-20) to genes that respond in Arabidopsis to bacterial challenge. Similarity, was much greater in the signaling (73% of ESTs with e values < 4e-20), cell growth and development (67% < e-43) and transport (88% < e-67) functional categories.
    • cDNA-AFLP analysis compared the transcriptional response of a resistant (G.41) and susceptible (M.26) cultivar. ESTs associated with resistance included proteins involved in perception, signaling, defense and apoptosis. Bioinformatics identified 30 ESTs (16%) that had not been reported for other stresses and appeared specific to the Malus- E. amylovora interaction.
  • ESTs were mapped to QTLs for fire blight resistance in Malus (in collaboration with Sue Gardiner, HortResearch, NZ).
    • ESTs were ranked for their potential impact on resistance based on bioinformatics and inferences drawn from model systems and the scientific literature.
    • SSR and SNP markers derived from highly ranked fire blight EST candidate genes were mapped in a M.9 X Robusta 5 population in which a major QTL for fire blight resistance has been located on Linkage Group 03. Candidate genes were mapped to this QTL, as well as to the positions corresponding to the location of at least two QTLs reported in other populations (Calenge et al. 2005, Khan et al. 2006).
    • These markers are being evaluated for their potential application in marker assisted breeding.
  • A high-efficiency transformation and selection system was developed to create apple RNAi silenced transgenics for determination of candidate gene function.
    • Thus far 15 candidate genes in six functional categories: general metabolism (1), photosynthesis (2), nucleic acid metabolism (1), protein metabolism (3), signaling (4), and defense/stress (4) have been subjected to this protocol.
Broad Impacts:

The genomic research undertaken in this project will elucidate the poorly understood mechanisms responsible for the resistance and susceptibility of apple and pear to fire blight disease and, thereby identify new opportunities for improving natural resistance.

  • The research project is identifying candidate fire blight resistance genes in apple and using RNAi to study the biological function of those genes.
  • It is also using candidate resistance genes to develop robust DNA markers for fire blight QTLs. This will facilitate new methods of marker-assisted selection to efficiently breed superior apple varieties with improved fire blight resistance resulting in:
    • reduced dependence on synthetic pesticides,
    • greater stability in production levels over time due to enhanced ability to better tolerate fire blight, and 3) improved competitiveness of the US fruit industry in the global market due to increased tree fruit productivity and reduced inputs.
Deliverables:
  • Publications:
    • Norelli, J.L., R.E. Farrell, Jr., C.L. Bassett, A.M. Baldo, D.A. Lalli, H.S. Aldwinckle, and M.E. Wisniewski. 2007/8. Rapid transcriptional response of apple to fire blight disease revealed by cDNA suppression subtractive hybridization analysis. Manuscript submitted to Tree Genetics and Genomes, July 3, 2007 Manuscript # TGG-2007-0056.
  • Presentations:
    • Borejsza-Wysocka, E.E., J.L. Norelli, M. Malnoy, R.E. Farrell, C.L. Bassett, H.S. Aldwinckle. 2007. Using a multi-vector transformation system for high-throughput generation of RNAi mutants of apple. Eucarpia Fruit Breeding Meeting, Zaragoza, Spain, Sept. 2007.
    • Lalli, D.A., T.S. Artlip, R.E. Farrell, Jr., M.E. Wisniewski, J.L. Norelli. 2007. Transgenic expression of Erwinia amylovora effectors eopB1 and hopCea in apple. 11th International Workshop on Fire Blight, Portland, OR. August, 2007.
    • Malnoy, M. , A. Baldo, E.E. Borejsza-Wysocka, J.L. Norelli, R.E. Farrell Jr., C.L. Bassett, H.S. Aldwinckle. 2007. Genes differentially expressed during interactions of resistant and susceptible apple cultivars with the Erwinia amylovora. Eucarpia Fruit Breeding Meeting, Zaragoza, Spain, Sept. 2007.
    • Malnoy, M. , A. Baldo, E.E. Borejsza-Wysocka, J.L. Norelli, R.E. Farrell Jr , C.L. Bassett, H.S. Aldwinckle. 2007. Differentially regulated genes determined by cDNA-AFLP analysis during interactions of Erwinia amylovora with resistant and susceptible apple cultivars. 11th International Workshop on Fire Blight, Portland, OR. August, 2007.
    • Norelli, J.L., R.E. Farrell, Jr., C.L. Bassett, A.M. Baldo, D.A. Lalli, H.S. Aldwinckle and M.E. Wisniewski. 2007. Rapid transcriptional response of Malus to Erwinia amylovora infection. 11th International Workshop on Fire Blight, Portland, OR. August, 2007.
  • Meeting Abstracts:
    • Baldo, A.J., C.L. Bassett, M. Malnoy, S.S Korban, K. Gasic, R.E. Farrell, H.S. Adlwinckle, J.L Norelli. 2007. Computational identification of candidate genes involved in response to fire blight in apples. PAG XV: http://intl-pag.org/15/abstracts/PAG15_P08c_886.html
    • Borejsza-Wysocka, E.E., J.L. Norelli, M. Malnoy, R.E. Farrell, C.L. Bassett, H. S. Aldwinckle. 2007. A multi-plasmid transformation approach for the high-throughput generation of RNAi mutants of apple. PAG XV: http://intl-pag.org/15/abstracts/PAG15_P05h_458.html
    • Farrell, R.E., Jr., C.L. Bassett, J.L. Norelli, A.Baldo, H.S. Aldwinckle, M.E. Wisniewski. 2007. Identification of genes expressed in apple (Malus X domestica) in response to Erwinia amylovora infection. PAG XV: http://intl-pag.org/15/abstracts/PAG15_P05h_467.html
    • Lalli, D.A., T.S. Artlip, R.E. Farrell, Jr., M.E. Wisniewski, J.L. Norelli. 2007. Chemically inducible expression of Erwinia amylovora bacterial effectors EopB1 and hopCea in apple. XIII International Congress on Molecular Plant-Microbe Interactions, July 21-27, 2007, Sorrento, Italy, Book of Abstracts, PS 14-747, page 343.
    • Malnoy M., E.E. Borejsza-Wysocka, R.E. Farrell, J.L. Norelli, C.L. Bassett, H.S. Aldwinckle. 2007. Differential apple gene expression during interactions of resistant and susceptible apple cultivars with the necrogenic bacterium Erwinia amylovora. PAG XV: http://intlpag. org/15/abstracts/PAG15_P05h_464.html
    • Norelli, J.L., C.L. Bassett, R.E. Farrell, Jr., A.M. Baldo, m. Malnoy, E. Borejsza-Wysocka, D.A. Lalli, S.S. Korban, K. Gasic, M.E. Wisniewski. 2007. Functional genomic response of apple to fire blight. PAG XV: http://intl-pag.org/15/abstracts/PAG15_P05h_465.html
  • Community resources generated:
    • Approximately 450 fire blight-associated Malus ESTs were deposited into GenBank
    • Approximately 6 molecular markers for fire blight QTLs
  • Training:
    • Undergraduates:
      • Michael P. O’Connor, undergraduate, Pennsylvania State University, York contributed to:
        • Norelli et al. publication in TGG
        • Norelli et al. presentation at 11th International Workshop on Fire Blight
    • Postdoctoral:
      • Dr. Donna A. Lalli, USDA-ARS, Kearneysville, WV; August 2006-present contributed to:
        • Lalli et al. presentation at 11th International Workshop on Fire Blight
        • Norelli et al. publication in TGG
        • Norelli et al. presentation at 11th International Workshop on Fire Blight
        • Lalli et al. meeting abstract for MPMI XIII
        • Norelli et al. meeting abstract for PAG XV
        • fire blight-associated Malus ESTs deposited into GenBank
        • molecular markers for fire blight QTLs (EST ranking)
Collaborations:
  • Established cooperative agreement between USDA-ARS and HortResearch, New Zealand to identify molecular markers for fire blight ESTs and determine if the markers are associated with QTLs for fire blight resistance. This has resulted in collaborations between the NRI project and Susan Gardiner (geneticist), Vincent Bus (plant breeder) and Erik Rikkerink (plant pathologist) at HortResearch, NZ.
  • Established a collaboration with Angela Baldo, USDA-ARS in bioinformatics research to:
    • custom-modify vector-screening tools to identify EST contamination, and
    • develop computational methods to identify apple genes expressed uniquely in disease-challenged tissue or that are similar to Arabidopsis ESTs associated with Pseudomonas syringae pv. tomato infection.
    • Established collaboration with Gennaro Fazio, USDA-ARS to facilitate vertical integration between NRI project and ARS/Cornell apple rootstock breeding program and to identify molecular markers for fire blight ESTs.
    • Bob Farrell obtained research support from NutriCore, N.E. and the York County Community Foundation to facilitate the study of the molecular biology of fire blight disease in apple. $6,000.00 received in 2007.