Progress 07/01/04 to 06/30/06
Outputs
Genetic strategies based on modulation of apple genes involved in disease development and resistance to disease in relation to fire blight were emphasized. 1) 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 had been transferred to Galaxy and M.26 apple genotypes. Several transgenic Galaxy lines had significantly increased resistance to fire blight in a growth chamber test. NPR1 protein levels were shown by western blot to be elevated in all MpNPR1 and AtNPR1 transgenic Galaxy lines. In lines with MpNPR1 driven by the inducible pin2 promoter the MpNPR1 protein level increased 2-3 fold after inoculation with Erwinia amylovora. 2) 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 a hairpin sequence
of DIPM1, were all transferred to Galaxy apple. RT-PCR showed that in some transgenic lines, one or more of the four DIPMs are fully or partially silenced. Many of these transgenic lines had significantly increased resistance to fire blight in growth chamber tests. Several of the resistant lines had significant silencing of 3 of the DIPMs. However some lines overexpressing DIPM1 also had increased resistance. Preliminary results showed that DIPM mRNA levels determined by RT-PCR are not a good predictor of DIPM protein levels in some lines. 3) In order to design another strategy to modulate gene expression of apple plants so that they would have greater resistance to fire blight, the interaction of apple proteins with the pathogenicity factor, HrpN, of E. amylovora was investigated. Interaction between harpin (HrpN) and harpin interacting protein (HIPM) was confirmed in an in vitro binding assay. To examine the role of both HIPM and AtHIPM, silencing constructs were designed. For this,
hairpin loop structures of both genes were constructed in pART27, a plant transformation vector. These were used to create 35 HIPM-silenced transgenic apples and AtHIPM-silenced transgenic Arabidopsis lines. The HIPM-silenced transgenic apple lines are now being evaluated for fire blight resistance. Some new synthetic pathogen-inducible plant promoters are being assayed for performance in apple. Preliminary analysis using the GUS gene showed similar expression to that by the CaMV35S promoter. For the commercialization of transgenic plants, use of a completely marker-free technology is desirable. We are developing a technique for apple transformation without any selectable marker. Transformation of the apple rootstock M.2 with the constructs pwiAtt35Sgus and pinMpNPR1 without the kanamycin resistance gene has been achieved. 1500 regenerants were harvested from leaf-piece transformation plates for each transformation. Between 250 and 300 were chosen randomly and tested by PCR for the
presence of the transgenes (attacin, GUS, NPR1, or the pin2 promoter). 22-25 percent of these regenerants showed integration of the transgene.
Impacts
This project will greatly improve the profitability of growing apples in those regions where the fire blight disease is prevalent. It will result in substantial reduction of the amount of antibiotics being sprayed in apple orchards, and will therefore lessen the frequency of antibiotic resistant bacteria in orchards. The rural economy of apple growing regions will be strengthened by increased profitability of apple farms, and the maintenance or addition of jobs and cash flow in their communities.
Publications
- Borejsza-Wysocki, W.S., Durst, R.A., and Aldwinckle, H.S. 2006. Immunoliposomes for early detection of Erwinia amylovora. Acta Hort. 704:63-68.
- Borejsza-Wysocka E.E., Malnoy M., Aldwinckle H.S., Meng, X., Bonasera, J.M., Nissinen, R.M., Kim, J.F. and Beer S.V., 2005. Increased resistance to fire blight in apple plants by silencing DspE-intercating proteins. Acta Hort. 704:509-513.
- LoGiudice, N., Aldwinckle, H.S., Robinson, T.L., and Fazio, G. 2006. The nature of resistance of the B.9 rootstock to fire blight. Acta Hort. 704:515-519.
- Malnoy M., Borejsza-Wysocka E.E., He S.Y., Aldwinckle H.S., 2005. An additional copy of the apple gene MpNPR1 in transgenic Malus X domestica induces increased disease resistance. Phytopathology 96:S180.
- Werner, EA and Aldwinckle, HS. 2005. A comparison of two years of research on biological control of fire blight in New York. Phytopathology 96:S183.
- Borejsza-Wysocka E.E., Malnoy M., Beer S.V., Aldwinckle H.S., 2005. Increased resistance to fire blight in apple plants by silencing DspE-intercating proteins. Phytopathology 96:S176.
- Malnoy M., Borejsza-Wysocka E.E., Aldwinckle H.S., Jin, Q.-L., and He, S.Y. 2006. Transgenic apple lines over-expressing the apple gene MpNPR1 have increased resistance to fire blight. Acta Hort. 704:521-526.
- Fazio, G., Aldwinckle, H.S., McQuinn, R.P., and Robinson, T.L. 2006. Differential susceptibility to fire blight in commercial and experimental apple rootstock cultivars. Acta Hort. 704:527-530.
- Malnoy M., Reynoird J.P., Borejsza-Wysocka E.E., Aldwinckle H.S., 2006. Activation of the pathogen-inducible Gst1 promoter of potato after elicitation by Venturia iaeaqualis and Erwinia amylovora in transgenic apple (Malus X domestica.). Transgenic Research. 15:83-93.
- Werner, NA, Heidenreich, G, and Aldwinckle, HS. 2005. Field evaluation of materials for control of fire blight infection of apple, 2004. Fungicide and Nematicide Tests 60:PF030.
- Dewdney, M.M., Seem, R.C., and Aldwinckle, H.S. 2005. Are they really there? Improving estimates of Erwinia amylovora populations in the MARYBLYTTM program. Phytopathology 95:S24.
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Progress 01/01/05 to 12/31/05
Outputs
Continuing last year's research, genetic strategies based on modulation of apple genes involved in disease development and resistance to disease in relation to fire blight were emphasized. 1) 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 had been transferred to Galaxy and M.26 apple genotypes. Several transgenic Galaxy lines had significantly increased resistance to fire blight in a growth chamber test. NPR1 protein levels were shown by western blot to be elevated in all MpNPR1 and AtNPR1 transgenic Galaxy lines. In lines with MpNPR1 driven by the inducible pin2 promoter the MpNPR1 protein level increased 2-3 fold after inoculation with Erwinia amylovora. 2) 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 a hairpin sequence of DIPM1, were all transferred to Galaxy apple. RT-PCR showed that in some transgenic lines, one or more of the four DIPMs are fully or partially silenced. Many of these transgenic lines had significantly increased resistance to fire blight in growth chamber tests. Several of the resistant lines had significant silencing of 3 of the DIPMs. However some lines overexpressing DIPM1 also had increased resistance. Preliminary results showed that DIPM mRNA levels determined by RT-PCR are not a good predictor of DIPM protein levels in some lines. 3) In order to design another strategy to modulate gene expression of apple plants so that they would have greater resistance to fire blight, the interaction of apple proteins with the pathogenicity factor, HrpN, of E. amylovora was investigated. Interaction between harpin (HrpN) and harpin interacting protein (HIPM) was confirmed in an in vitro binding assay. To examine the role of both HIPM and AtHIPM,
silencing constructs were designed. For this, hairpin loop structures of both genes were constructed in pART27, a plant transformation vector. These were used to create 35 HIPM-silenced transgenic apples and AtHIPM-silenced transgenic Arabidopsis lines. The HIPM-silenced transgenic apple lines are now being evaluated for fire blight resistance. Some new synthetic pathogen-inducible plant promoters are being assayed for performance in apple. Preliminary analysis using the GUS gene showed similar expression to that by the CaMV35S promoter. For the commercialization of transgenic plants, use of a completely marker-free technology is desirable. We are developing a technique for apple transformation without any selectable marker. Transformation of the apple rootstock M.26 with the constructs pwiAtt35Sgus and pinMpNPR1 without the kanamycin resistance gene has been achieved. 1500 regenerants were harvested from leaf-piece transformation plates for each transformation. Between 250 and 300
were chosen randomly and tested by PCR for the presence of the transgenes (attacin, GUS, NPR1, or the pin2 promoter). 22-25 percent of these regenerants showed integration of the transgene.
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
- Malnoy M., Borejsza-Wysocka W., Jin Q.L., He S.Y., Aldwinckle H.S. 2005. Resistance to Erwinia amylovora in apple through over-expression of the Malus x domestica gene NPR1. XII International Congress on Molecular Plant-Microbe Interactions, 14-18 December, 2005, Cancun, Mexico.
- Borejsza-Wysocka E., Malnoy M., Meng M., Bonasera J.M., Beer S.V., Aldwinckle H.S. 2005. Silencing of DspE interacting proteins increases resistance to fire blight of susceptible apple genotypes. XII International Congress on Molecular Plant-Microbe Interactions, 14-18 December, 2005, Cancun, Mexico.
- Malnoy M., Borejsza-Wysocka E.E., He S.Y., Aldwinckle H.S. 2005. An additional copy of the apple gene MpNPR1 in transgenic Malus X domestica induces increased disease resistance. 65th annual meeting of American Phytopathology Society North-eastern division. 5-7 October, 2005, Geneva, USA.
- Aldwinckle H.S., Malnoy M., Borejsza-Wysocka E.E., Abbott M.J., Lewis S.A., Norelli J.L., Beer S.V. and He S.Y. 2005. Using rDNA technology to obtain fire blight resistance in apple fruit cultivars. 65th annual meeting of American Phytopathology Society North-eastern division. 5-7 October, 2005, Geneva, USA.
- Borejsza-Wysocka E.E., Malnoy M., Beer S.V., Aldwinckle H.S. 2005.Increased resistance to fire blight in apple plants by silencing DspE-intercating proteins. 65th annual meeting of American Phytopathology Society North-eastern division. 5-7 October, 2005, Geneva, USA.
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Progress 01/01/04 to 12/31/04
Outputs
Genetic strategies based on modulation of apple genes involved in disease development and resistance to disease in relation to fire blight were emphasized. 1. 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. 2. 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 a hairpin 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. 3. In order to design another strategy to modulate gene expression of apple plants so that they would have greater resistance to fire blight, the interaction of apple proteins with the pathogenicity factor, HrpN, of Erwinia amylovora was investigated. Interaction between harpin (HrpN) and harpin interacting protein (HIPM) was confirmed in an in vitro binding assay. T7-tagged HIPM and HrpN proteins were mixed with synthetic beads that had been conjugated with T7 tag antibody. After the putative complex was released from the beads, HrpN was detected in the complex by western blotting with an
anti-HrpN antibody. Anti-T7 tag antibody was used to check for the presence of T7-tagged HIPM protein. HrpN was pulled down only in the presence of HIPM, indicating that HIPM interacts with HrpN at the protein level. Based on the amino acid sequence of HIPM, orthologs called 'AtHIPM', from an Arabidopsis database were found and cloned from the Columbia and WS ecotypes of Arabidopsis by RT-PCR methodology. The two AtHIPMs were 100 percent identical at the amino acid level, and they were about 70 percent identical to HIPM. In yeast, AtHIPM interacted with HrpN just like HIPM. This means that the HrpN target likely is present not only in host plants like apple, but also in non-host plants like Arabidopsis. To examine the role of both HIPM and AtHIPM, silencing constructs were designed. For this, hairpin loop structures of both genes were constructed in pART27, a plant transformation vector. These are being used to create HIPM-silenced transgenic apples and AtHIPM-silenced transgenic
Arabidopsis lines.
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
- No publications reported this period
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