Progress 07/01/02 to 06/30/03

Outputs
In spring 2002 flowers of transgenic apple trees under bee-exclusion netting were pollinated with Idared pollen. On 9 Sept 2002, all transgenic fruit, along with fruit of non-transgenic Royal Gala and Galaxy from the same rows were harvested, stored at 2C, and subsequently graded for size and color. In May 2003 flowers were again manually pollinated. In 2002, 3- to 6-yr-old plants of Royal Gala, Galaxy, and M.26 transgenics containing various lytic proteins (attacin E, cecropin SB-37, and avian and T4 phage lysozymes) were evaluated for their fire blight resistance by inoculating shoot tips with the fire blight bacterium, measuring the extent of the resulting infection, and comparing it with that of non-transgenic Royal Gala, Galaxy or M.26 parents. 5 to 10 plants of each line had been planted in a completely randomized design and up to 15 shoots were inoculated per plant. An improved lytic protein construct including the wound inducible proteinase inhibitor II promoter from potato (Ppin2), a translational enhancer from alfalfa mosaic virus (AMV), and a signal peptide sequence from the pathogenesis related protein 1b of tobacco (SP) was transferred to Galaxy and M.26. Transgenic plants were propagated in vitro and gene transfer confirmed by molecular methods. The expression of lytic protein has been characterized, and all lines were tested for resistance in the growth chamber. They were then be propagated in the growth chamber and greenhouse and planted in the field in spring 2003. The HrpN gene was transferred into apple to determine whether it would produce harpin protein in apple plants and make them more resistant to fire blight. Tests indicated that harpin is produced in transgenic apple plants. A growth chamber test indicated that resistance was significantly increased in transgenic M.26 plants containing the Pgst1-SS-hrpN construct. This was supported by a preliminary field test of resistance on a few plants. The apple gene for NPR1 protein (MpNPR1), which is a critical factor in disease resistance in Arabidopsis and rice, was isolated by S. He, Michigan State University. MpNPR1 driven by Ppin2 was transferred to Galaxy and M.26. Several transgenic Galaxy lines had significantly increased resistance to fire blight in a growth chamber test. It was shown that four kinase proteins in apple are necessary to interact with the DspE protein produced by Erwinia amylovora, in order for infection to develop. To silence the four DspE-Binding kinases (DIPMs) in apple, constructs with short sequences of each gene as well as a construct with all 4 short sequences together, a complete sequence, and an RNAi sequence of DIPM1, were all transferred to Galaxy apple. Plants of all transgenic lines have now been propagated, and tested for gene expression. We have shown that in some transgenic lines, one or more of the four DIPMs are fully or partially silenced. Plants of many of these transgenic lines have also been evaluated for fire blight resistance in preliminary tests in the growth chamber. Five lines showed significantly increased resistance. Three of those 5 lines had significant silencing of 3 of the DIPMs.

Impacts
Our research has shown clearly that it is feasible to greatly improve the resistance of good apple varieties, like Gala, to the devastating fire blight disease by adding genes using biotechnology. We have shown that we can use a gene from the bacteria that cause the disease to make the apple plant resistant to it. In addition, apple genes, taken from apple plants, are now being enhanced to increase resistance. These discoveries will lead to new fire blight resistant strains of our best apple varieties and rootstocks that should be readily accepted by growers and consumers alike. This will result in savings for growers because of reduced losses due to fire blight and reduced spray costs. It will also be beneficial to the environment because antibiotic and copper sprays will no longer be needed.

Publications

• Ko, K., Norelli, J. L., Aldwinckle, H. S. and Brown, S. K. 2002. T4 lysozyme and attacin genes enhance resistance of transgenic 'Galaxy' apples against Erwinia amylovora. Journal of American Society for Horticultural Science 127: 515-519.
• Aldwinckle, H. S., Borejsza-Wysocka, E.E., and Norelli, J.L. 2002. Quality of fruit of lytic protein transgenic apple lines with enhanced resistance to fire blight. Congress of International Association for Plant Tissue Culture and Biotechnology. Orlando, FL. June 2002.
• Aldwinckle, H. S. 2002. Development of fire blight resistant apple cultivars by genetic engineering; present and future. International Horticulture Congress, Toronto, Canada, August 2002.
• Aldwinckle, H. S. 2002. Recent advances in genetic engineering of apple. First International Symposium on rootstocks for deciduous fruit tree species. Zaragoza, Spain, 11-14 June 2002

Progress 01/01/02 to 12/31/02

Outputs
Excellent crops were obtained on transgenic Royal Gala and Galaxy trees in 2001. These included Royal Gala lines transgenic for attacin, avian lysozyme, and SB37 cecropin, and Galaxy lines transgenic for attacin and viral lysozyme. As required by our USDA-APHIS permit, flowering trees were bee-netted during bloom, and manually pollinated. All transgenic fruit, along with fruit of normal Royal Gala and Galaxy from the same rows, were graded for size and color, and will be sampled for firmness, soluble solids and titratable acidity. Results indicate that the quality and varietal characteristics of transgenic Royal Gala and Galaxy are not different from those of the parent varieties. Field trials of the resistance to fire blight of Royal Gala, Galaxy, and M.26 rootstock transgenic lines containing lytic proteins continued on older plants and were begun on young plants. Resistance to fire blight caused by the lytic protein genes continues to remain stable as trees age. Some transgenic lines of the very susceptible M.26 appear to have increased resistance, and may be of commercial interest. Results of this project to date show clearly that genetic engineering can be applied to significantly increase the resistance of apple varieties to fire blight, without altering their desirable varietal characteristics. However because of regulatory, intellectual property, and public acceptance concerns about the lytic protein genes first used in this project, commercialization of the first transgenic lines will be lengthy and costly. To mitigate these concerns, several years ago we began work with other types of genes as they became available, from research at Cornell and Michigan State Universities. Most progress has been made with the harpin gene from the fire blight bacterium. Transgenic lines of M.26 with this gene have shown increased resistance in growth chamber and preliminary field trials. Many transgenic lines have been produced and are being propagated for resistance tests. Harpin transgenic lines of Galaxy are also being produced. A second type of gene, MpNPRI, which is from apple itself, and is thought to be responsible for disease resistance in apple, has been transferred to Galaxy so that it will be expressed at a higher level than normal and therefore increase resistance. A third new technology is based on silencing proteins in the apple that are needed by the fire blight bacterium to cause disease. Seven different silencing constructs are now being transferred to Galaxy. Field trials of new control materials for use in orchards of susceptible varieties have shown that S-0208 (oxolinic acid) and Phyton 27 are very effective against blossom blight, but do not yet have registration in the U.S. Apogee is very effective against shoot blight when applied at the appropriate rate considering the vigor of the trees and the seasonal growing conditions. It is registered in the U.S. and is expected to play a major role in shoot blight control on mature trees in the future. Its use on immature trees requires further study. Serenade and Messenger are registered in the U.S. but require further testing to better understand their effectiveness.

Impacts
A biological material containing a benign bacterium has given promising results and is now registered for growers to trial. Other biological materials have also been tested, as well as versions of older chemicals that would be usable in organic production. For the future, we have developed new varieties and rootstocks of apple for growers to plant in new orchards. We have used both traditional breeding and biotechnology to do this. Already several resistant rootstocks have been developed by breeding and are starting to be used on a trial basis by growers. Resistant apple fruit varieties have been produced by biotechnology, and have been shown to have stable resistance and to have normal fruits. Versions of these varieties suitable for commercial use are now being developed. They will use improvements in certain genes that are already present in apple plants, and will therefore present no concerns for the environment or health. Since we are still developing most of these methods for improving management of fire blight, the impact of our work still lies largely in the future. However growers have already started to use some of the fire blight resistant rootstocks that we have bred. The rootstocks will greatly reduce, or even eliminate death of trees due to infection of the roots by fire blight. It is also expected that some growers will use the biological material to reduce infection of flowers this year. Besides reducing crop losses, use of this material instead of streptomycin antibiotic will reduce the chance of streptomycin resistance arising in orchards.

Publications

• No publications reported this period