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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
Utilize functional genomic/proteomic approaches to identify genes or proteins in fruit crops that confer resistance or susceptibility to freezing or water stress and fire blight. Develop and evaluate new transgenic technologies needed to control gene flow and expression in transgenic apple trees. Evaluate genetically engineered rootstocks as (1) a means of enhancing resistance to diseases and (2) a means of indirectly influencing gene expression in the scion for the improvement of performance or quality.

1b.Approach (from AD-416)
Transcript profiling will be used to identify genes associated with environmental stress, fire blight resistance, and susceptibility, while proteomic approaches will be used to better understand the relationship between gene expression and cognate protein levels. RNAi-induced gene siliencing will be used to elucidate the role of specific candidate genes in resistance and adaptation. The ability to use genetically-engineered rootstocks of apple for scion trait modification will be explored by investigating graft-transmissible gene silencing. The use of floral-specific promoters to confine and regulate the expression of dehydrin genes (responsible for cold and dehydration tolerance) to flowers of fruit crops in order to improve frost tolerance will also be investigated.

3.Progress Report
Plants are immune to infection by most micro-organisms and in order to cause disease pathogens must first suppress or overcome natural plant immunity. Using a microarray representing 39,000 apple genes we identified 1,541 genes involved in apple disease immunity following challenge with bacterial plant pathogens, including the fire blight pathogen. This subset of 1,541 genes are now being analyzed to identify candidate fire blight resistance genes and to develop molecular markers for use in marker assisted breeding of fire blight resistant apple cultivars.

A candidate fire blight resistance gene is located within a known genetic locus for fire blight resistance in the wild apple cultivar 'Robusta 5'. This gene was cloned from 'Robusta 5', fire blight resistant apple rootstock 'Geneva 41', and susceptible rootstocks 'Malling 26' and 'Malling 27'. Four variants, or alleles, of the gene were identified and one of the variants was present in both resistant 'Robusta 5' and 'Geneva 41', suggesting it may be the gene responsible for resistance. We are now using transformation technologies to over-express the gene variants in a susceptible apple cultivar and determine if this gene plays a causative role in fire blight resistance.

To complement a previous gene expression study using expressed sequence tags (ESTs), we performed suppression subtractive hybridization (SSH) to identify weakly expressed genes in apples subjected to low temperature (1 and 24 h at 4 degrees C) and 2 weeks of drought. We are continuing to clone and sequence the subtracted sequences from the bark tissue subjected to drought treatment.

An analytical verification of 6 flower-specific constructs containing a flower-specifc promoter from petunia (pZPT) driving expression of single and combinations of peach dehydrins is near completion. The six contructs have been transformed into Arabidopsis thaliana in order to evaluate their ability to protect the female flower part from frost damage. Multiple lines of each of the 6 constructs have been obtained and have been evaluated by PCR to verify the presence and integrity of the constructs. Evaluation of the dehydrin protein levels in the transformed lines is being conducted using antibody techniques.

Several lines of transgenic apple overexpressing a native CBF transcription factor have been evaluated for cold hardiness. Since CBF regulates the expression of genes associated with cold resistance in other plant species, it was expected that similar results would be obtained in apple. At least 3 independent lines have been evaluated and we have demonstrated that increased levels of cold hardiness are present in all the lines and that the level of increase is directly related to the level of expression of the CBF genes. Overexpression of the CBF genes was also associated with increased sensitivity to dormancy cues and with reduced growth. An apple microarray has been employed to identify what genes are being regulated by CBF in apple.

1. Fire Blight Pathogen Suppresses Resistance to Help Infect Fruit Trees. Fire blight is a destructive disease of apple and pear trees that is estimated to cost the US fruit industry over $100 million a year in crop losses and disease control. It is known that the fire blight pathogen uses a secretion complex to deliver "effector" proteins into apple/pear cells that cause disease, however the biological function of most fire blight effectors is not known. ARS scientists constructed plant vectors to express the fire blight Eop1 effector protein in apple and found that it suppressed natural plant immunity that would normally inhibit microbial colonization, thus facilitating infection by the pathogen. Understanding the mechanisms by which the fire blight pathogen regulates defense responses in apple and pear will facilitate the development of natural host plant resistance to manage this devastating fruit tree disease.

2. The physical structure of DNA changes in response to cold temperature is predicted. Frost and freeze damage results in significant yearly losses in fruit yields and shortens tree longevity. All of the responses a fruit tree makes to defend itself against cold temperature extremes have not yet been discovered. Although changes in gene expression that are associated with low temperature have been well documented, only a few factors involved with regulating those genes have been identified. Using bioinformatic software that predicts changes in nucleic acid secondary structure, we discovered that regulatory regions in two cold-responsive dehydrin genes were predicted to change shape dramatically below 15 degrees C, while a third dehydrin unresponsive to cold showed no change in its shape from 25 degrees C to 0 degrees C. This observation is the first to suggest that physical changes in DNA at regulatory regions could contribute to a tree's ability to respond to cold. Using this information we can design regulatory regions with similar structures in other cold-responsive genes to boost their expression during exposure to low temperature extremes and improve the tree's chance of survival.

6.Technology Transfer

Number of the New/Active MTAs (providing only)1

Review Publications
Gasic, K., Yuepeng, H., Kertbundit, S., Shulaev, V., Lezzoni, A., Stover, E.W., Bell, R.L., Wisniewski, M.E., Korban, S. 2009. Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species. Molecular Breeding. 23:397-411.

Gasic, K., Gonzalez, D.O., Thimmapuram, J., Malnoy, M., Gong, G., Han, Y., Vodkin, L.O., Lei, L., Aldwinckle, H.S., Carroll, N., Orvis, K., Goldsbrough, P., Clifton, S., Pape, D., Fulton, L., Martin, J., Theising, B., Wisniewski, M.E., Fazio, G., Korban, S.S. 2009. Comparative analysis and functional annotation of a large expressed sequence tag collection of apple. The Plant Genome. 2(1):23-38.

Bassett, C.L., Wisniewski, M.E., Artlip, T.S., Richart, G., Norelli, J.L., Farrell, Jr., R.E. 2009. Comparative expression profiling and transcript initiation of three peach dehydrin genes. Planta. 230:107-118.

Peace, C., Norelli, J.L. 2009. Genomics approaches to crop improvement in the Rosaceae. In: Folta, K.M., Gardiner, S.E., editors. Genomics of the Rosaceae. Springer. p. 19-54.

Last Modified: 8/27/2015
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