Source: AGRICULTURAL RESEARCH SERVICE submitted to
MOLECULAR GENETIC APPROACH FOR IMPROVED PATHOGEN AND PEST RESISTANCE IN SUGAR BEET
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0406011
Grant No.
(N/A)
Project No.
1275-21220-183-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 14, 2002
Project End Date
Apr 11, 2006
Grant Year
(N/A)
Project Director
SMIGOCKI A C
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2042010106010%
2062010104010%
2112010104030%
2112010106030%
2122010104020%
Goals / Objectives
1. Characterize defense response genes in sugar beet in order to develop effective strategies for control of diseases and pest infestations in plants. Determine the effect of a cytochrome P450 gene on insect and disease resistance. 2. Develop strategies to genetically improve Cercospora leafspot resistance based on the manipulation of previously characterized disease resistance genes. 3. Devise sugar beet maggot control strategies based on expression of proteinase inhibitor genes targeting the maggot's major digestive midgut proteases.
Project Methods
Profile taproot genes expressed in root maggot resistant vs. susceptible sugar beet varieties to identify genes that comprise the resistance mechanism response. Profile genes expressed in response to Cercospora in sugar beet using both highly susceptible and moderately resistant genotypes. Incorporate into the genomes of sugar beet and/or spinach the biofungicide genes from Cercospora-antagonistic Pseudomonas bacteria, cfp toxin export pump gene from Cercospora, Arabidopsis npr1 gene, heterologous digestive proteinase inhibitor genes, or the cytochrome P450 enzyme gene involved in the synthesis of insecticidal secondary metabolities. BL-1; recertified January 27, 2003.

Progress 09/14/02 to 04/11/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Agronomic losses due to disease-producing bacteria, fungi and insects can be reduced by the genetic construction of transgenic plants carrying genes for the production of antimicrobial and insecticidal proteins and compounds. Environmentally friendly means of controlling disease producing microbes and insects are needed in order to reduce use of chemical pesticides in farming. Sugar production from sugar beet has not increased for the last 30 years mainly due to pathogenic microbes and insect pests. The most important insect pest of sugar beet in United States is the sugar beet root maggot (Tetanops myopaeformis). The root maggot has become established at economically damaging levels in more than half of the more than 1.5 million acres grown annually, causing 10 to 100% reduction in yield. Cercospora-induced leafspot and Erwinia- induced root rot are major microbial diseases affecting the profitability and sustainability of sugar beet production in most areas where the crop is grown. Estimates of the economic losses vary from 20 to 30% and in some years could even be higher. Chemical control of sugar beet and other plant diseases and pests is becoming less effective due to the increasingly widespread occurrence of resistance. In addition, the EPA is re-evaluating the safety of some chemical agents whose future availability for control of sugar beet pests and pathogens is uncertain. The introduction of reconstructed beneficial genes that specifically target sugar beet pests and pathogens offers a novel means to augment plant breeding to confer disease and insect resistance to an important crop plant. Specific goals of the project are to develop strategies to genetically improve Cercospora leafspot and root maggot resistance based on the manipulation of previously characterized disease resistance genes from heterologous systems and newly discovered sugar beet resistance genes. We have identified and reconstructed a number of beneficial genes for specific expression in sugar beet. We are also developing approaches to identify disease resistance genes in moderately resistant sugar beet varieties. Characterization of the genes will lead to development of novel non-chemical strategies for effective and safe control of sugar beet diseases and pests. This research falls under National Program 302, Plant Biological and Molecular Processes, Component 2B, Understanding Plant Interactions with Their Environment. This project will increase the knowledge and understanding of the basis for resistance to pests and pathogens of a major US crop, sugarbeet. This research will provide 1) knowledge of the regulatory mechanisms that govern plant gene expression; 2) technologies for controlling gene expression to achieve inherent increased productivity and quality characteristics in plant germplasm; and 3) methodology to enhance the development of improved elite germplasm lines with enhanced disease and pest resistance that will lead to increases in yields and the quality and nutritional value of cultivated crops. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY2002) 1. Identify known genes with potential for improving Cercospora leafspot resistance in sugar beet, Beta vulgaris L. 2. Develop improved transformation and regeneration protocols for sugar beet, specifically so that transgenic plants can be readily constructed. 3. Determine the feasibility of developing a laboratory bioassay for the sugar beet root maggot using sugar beet seedlings and spinach as a model plant for sugar beet. 4. Initiate the collection of taproot tissues from root maggot infested sugar beet lines ( a moderately resistant and a corresponding parental susceptible line) for preparation of cDNA libraries enriched for genes associated with insect and disease resistance. 5. Transform model plants with a reconstructed cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 2 (FY2003) 1. Construct transgenic sugar beet plants carrying the CFP cercosporin transport gene from Cercospora. 2. Verify the genomic insertion of the introduced CFP gene in transgenic sugar beet using PCR with gene-specific primers. 3. Determine the feasibility of developing a root maggot bioassay using sugar beet hairy root cultures derived from moderately resistant and susceptible sugar beet lines for future use as tools for rapid evaluation of newly characterized disease resistance genes. 4. Initiate preparation of cDNA libraries of taproot tissues collected at specific time points following infestation with the root maggot larvae to preferentially select insect and disease resistance genes. 5. Initiate molecular analysis of genetically modified plants transformed with a cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 3 (FY2004) 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. 3. Initiate a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotova. 4. Devise specific primers for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. 5. Introduce a reconstructed reporter gene (GUS) into hairy roots for analysis of tissue-specific and constitutive gene expression in sugar beet root cells. 6. Continue to prepare cDNA libraries from root maggot infested sugar beet tissues for cloning and characterization of disease resistance- associated genes using a subtractive enrichment approach. 7. Molecularly reconstruct proteinase inhibitor genes that specifically target the root maggot digestive proteases for expression in sugar beet hairy root cultures and spinach plants (model for sugar beet) to evaluate the genes effects on root maggot control. 8. Determine cytochrome P450 protein levels in genetically engineered model plants and relate P450 gene expression to pest and pathogen resistance. Year 4 (FY2005) 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. Begin planting seed from crosses in greenhouses (Beltsville, MD) for evaluation next year. 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. 5. Initiate preparation of sugar beet macroarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures for root maggot resistance analysis using the newly developed root maggot bioassay. 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Year 5 (FY2006) 1. Evaluate Cercospora resistance levels of transgenic plants crossed with agronomically elite germplasms using an artificial field inoculation procedure in the growth chamber. 2. Analysis of genes controlling pathogenesis in the species-specific root rot- causing bacterial phytopathogen (Erwinia betavasculorum) using site direced mutagenesis. 3. Sequence analyses of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. 4. Screen sugar beet macroarrays for genes whose expression is modulated increased or decreased) by the interaction with the root maggot using molecular probes prepared from sugar beet lines that are moderately resistant and susceptible to the root maggot. 5. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures and spinach plants. 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. 4a List the single most significant research accomplishment during FY 2006. To improve root maggot and Cercospora resistance in sugar beet using biotechnological approaches, we needed to identify disease resistance genes and to develop pathogen and insect bioassays for screening newly developed plant varieties for resistance. To characterize sugar beet resistance genes, we prepared libraries (cDNAs) enriched for root maggot resistance genes from moderately resistant and susceptible breeding lines that were developed by ARS scientists in Fargo, ND in collaboration with scientists at North Dakota State University. Among the differentially expressed genes, those with potential roles in sugar beet defense mechanisms were identified for further characterization. Selected genes were reconstructed for over-expression in plants and reconstructed genes were introduced into sugar beet roots (in vitro propagated hairy root cultures). Insect bioassays were developed with the regenerated hairy root cultures in order to quickly identify genes that have the potential to improve pest and disease resistance, thus leading to increased sugar yields and reduction in pesticide usage. A bacterial artificial chromosome (BAC clone) which carries the disease and pest resistance control gene NPR1 was identified in a screen of a genetic library of sugar beet Hybrid US H2O in collaboration with ARS, East Lansing, MI. The identified BAC clone was sequenced and decoded and a 38,500 base contiguous segment containing the NPR1 homologous sequence was submitted to GenBank, accession DQ851167. This new scientific information will be useful to us and other molecular geneticists who are working to improve disease and pest resistance in crop plants. This research is related to NP302, Component 2B. 4b List other significant research accomplishment(s), if any. Viable seed were obtained from genetic crosses of the CFP transgenic T7 with agronomically elite biennial germplasm constructed in Salinas. Seed was received from our ARS collaborators at Fort Collins, CO and about 30 plants are being propagated for PCR confirmation and Cercospora leaf spot susceptibility evaluation. In a new genetic study of pathogenesis/virulence in Erwinia betavasculorum, motility and chemotaxis were found to be avirulence factors which are presumed to induce host defense mechanisms and interfere with pathogenesis. Virulent mutants were selected by plant interaction and shown to be impaired in both motility and chemotaxis. We devised specific gene markers (DNA primers) useful for identifying sugar beet genes homologous to the disease resistance-determining gene of Arabidopsis (NPR1) and these have preliminarily been used to identify large stretches of sugarbeet DNA (BAC clones) that may contain the sugar beet NPR1 gene. Characterization of the genes controlling mechanisms may be useful for increasing disease and pest resistance in sugar beet. Identified clones associated with root resistance mechanisms; cloned sugar beet root genes that are regulated by root maggot feeding from roots of moderately resistant and susceptible sugarbeet varieties. (manuscript published) Sugar beet was transformed with proteinase inhibitor (PI) genes that target the sugar beet root maggot digestive enzymes. Nine promoter-PI gene transformation vectors were constructed in strategically designed vector cassettes. Cassettes were developed to facilitate rapid cloning of newly discovered resistance genes and regulatory switches for analysis of gene function in root maggot susceptible and moderately resistant sugar beet germplasm. Molecular analyses of transformed hairy root cultures were initiated to determine levels of expression of PI genes for subsequent evaluation of insect resistance. Insect bioassays were developed utilizing in vitro propagated sugar beet hairy root cultures. A number of sugar beet pests were evaluated (beet fall armyworm; sugar beet root aphid) as well as other pests (Southern corn root worm, tobacco bud worm). Using hairy roots as a model system, we studied the interaction between sugarbeet roots and the sugarbeet root maggot that is important for designing novel control approaches that disrupt this interaction. In addition, studies were initiated to evaluate the effects of newly identified resistance genes and toxic compounds on sugarbeet pests and pathogens that specifically target the taproot and rhizosphere. We demonstrated that the expression of a defense-related cytochrome P450 gene in transgenic plants enhanced resistance to insects and pathogenic microbes. The level of gene expression was correlated to the levels of observed disease resistance. Leaf extracts from resistant plants were shown to be insecticidal activity and are in the process of further analysis to identify the active compound(s). This research is related to NP302, Component 2B. 5. Describe the major accomplishments to date and their predicted or actual impact. The CFP gene from Cercospora was identified as a good candidate gene for improving Cercospora leaf spot resistance in sugar beet. Sugar beet was transformed with the CFP gene and a transgenic plant carrying the CFP gene was regenerated. Progeny plants were crossed with agronomically improved, elite germplasm and the resulting viable progeny are undergoing evaluation as to the value of these genetic materials for incorporation into Cercospora leaf spot resistance breeding programs. The question being addressed is whether the CFP gene can be recombined into relatively resistant germplasm in order to effectively raise the level of Cercospora leaf spot resistance. Established in vitro methodology for generating transformed sugar beet hairy root cultures to facilitate rapid evaluation of the effects of transgenes on pest and disease resistance. An in vitro sugar beet root maggot bioassay was developed using both sugar beet seedlings and transformed hairy root cultures that will facilitate rapid screening of newly developed sugar beet germplasm for resistance to the root maggot, the most devastating insect pest of sugar beet, and potentially other sugar beet pests and pathogens. We developed improved gene transfer methodology for sugar beet in order to facilitate the introduction of beneficial genes into sugar beet for improved disease and insect resistance. We developed a method to transform embryogenic sugar beet suspension cultures with reporter genes using the biolistic transformation method. We optimized the generation of embryogenic leaf callus from commercially important sugar beet lines for use with the biolistic transformation method and successfully regenerated transgenic plants that express an introduced transgene. Sugar beet taproot cDNA libraries enriched for defense response genes were prepared and will facilitate the identification and characterization of genes involved in defense mechanisms. Biotechnological strategies to control diseases and pests will be designed based on the modulation of expression of these defense related genes in plants. Provided the scientific community with over 450 sugar beet root ESTs (GenBank accession numbers DV501516 through DV501974) that are associated with root defense response mechanisms. Plant transformation vectors were constructed for use as cassettes that allow for rapid cloning of newly discovered resistance genes and regulatory switches for subsequent introduction into plants for analysis of gene function and expression. We developed genetically modified plants with a reconstructed gene that is involved in the synthesis of plant compounds that have insecticidal and pharmaceutical properties. Model plants expressing the defense related gene were resistant to insects and microbial pathogens leading to the possibility of utilizing this gene in other plants. We demonstrated that the expression of a defense-related cytochrome P450 gene in transgenic plants enhanced their resistance to insects and pathogenic microbes. Levels of expression of the P450 gene in transgenic plants were determined in order to correlate them with the observed levels of disease resistance. Insecticidal compounds were localized to the leaf surfaces by preparation and analysis of leaf extracts (manuscript published). Collaborative efforts are being explored to purify and identify the active compound(s). This research is related to NP302, Component 2B. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Research progress is documented in annual reports to the Beet Sugar Development Foundation and the American Society of Sugar Beet Technologists (ASSBT). A CRADA was continued for exploring the production of renewable biomass fuel from sugar beet that ultimately would benefit the sugar beet grower and processor industries. A TRUST agreement was continued for identifying disease resistance traits to improve sugar beet for industry and processor groups. An SCA with a university collaborator is ongoing to further characterize cloned plant genes induced by feeding insects that will be developed for use as potential targets for pest control. Sugar beet DNA sequences containing valuable DNA sequence and gene annotation information were submitted to the GenBank database for online access by all scientists interested in improvement of disease and pest resistance in plants. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Kuykendall, L.D., Panella, L., Lewellen, R.T. 2006. Progeny from genetic crosses of CFP transgenic T7#12 with two Salinas genotypes. Sugarbeet Research 2005 Report, pp. E7-E13. Smigocki, A. 2006. Cloning of Beta vulgaris root ESTs modulated by sugar beet root maggot feeding: role of proteinase inhibitors in insect resistance. Beet Sugar Development Foundation Res. Rep. E2-6. Smigocki, A. 2006. Sugar beet root genes modulated by sugar beet root maggot (SBRM) feeding: role of proteinase inhibitors in insect resistance. Sugarbeet Research Executive Summary, 2005 Report, Beet Sugar Development Foundation.

Impacts
(N/A)

Publications

  • Puthoff, D.P., Ivic-Haymes, S., Zuzga, S., Smigocki, A.C. 2006. Beta vulgaris root ests modulated by sugar beet root maggot feeding: their regulation by wounding and defense signals and potential role for insect control. National Academy of Sciences Sackler Symposium, From Functional Genomics of Model Organisms to Crop Plants for Global Health. p. 36.
  • Puthoff, D.P., Smigocki, A.C. 2006. Beta vulgaris root ests modulated by sugar beet root maggot feeding and their regulation by wounding and defense signaling molecules. Plant Cell Rep., online first (DOI 10. 1007/s00299-006- 0201-y).
  • Kuykendall, L.D., Panella, L.W., Lewellen, R.T. 2006. Progeny of genetic crosses of CFP transgenic sugarbeet with two salinas genotypes. Sugarbeet Research and Extension Reports. pp. E7-E13.
  • Smigocki, A.C. 2006. Cloning of beta vulgaris root ests modulated by sugar beet root maggot feeding: role of proteinase inhibitors in insect resistance. Annual Beet Sugar Development Foundation Research Report. Res. E2-6.
  • Puthoff, D.P., Ivic-Haymes, S., Zuzga, S., Smigocki, A.C. 2006. Beta vularis root defense responses: root ests regulated by the sugar beet root maggot and their potential role in insect resistance. BARC Poster Day. p. 40.
  • Smigocki, A.C., Ivic-Haymes, S.D., Campbell, L.G., Boetel, M.A. 2006. Screening beta vulgaris l. for sugarbeet root maggot (tetanops myopaeformis roder) resistance using an in vitro bioassay. Journal of Sugarbeet Research. 43:1-13.


Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Agronomic losses due to disease-producing bacteria, fungi and insects can be reduced by the genetic construction of transgenic plants carrying genes for the production of antimicrobial and insecticidal proteins and compounds. Environmentally friendly means of controlling disease producing microbes and insects are needed in order to reduce use of chemical pesticides in farming. Sugar production from sugar beet has not increased for the last 30 years mainly due to pathogenic microbes and insect pests. The most important insect pest of sugar beet in United States is the sugar beet root maggot (Tetanops myopaeformis). The root maggot has become established at economically damaging levels in more than half of the more than 1.5 million acres grown annually, causing 10 to 100% reduction in yield. Cercospora-induced leafspot and Erwinia- induced root rot are major microbial diseases affecting the profitability and sustainability of sugar beet production in most areas where the crop is grown. Estimates of the economic losses vary from 20 to 30% and in some years could even be higher. Chemical control of sugar beet and other plant diseases and pests is becoming less effective due to the increasingly widespread occurrence of resistance. In addition, the EPA is re-evaluating the safety of some chemical agents whose future availability for control of sugar beet pests and pathogens is uncertain. The introduction of reconstructed beneficial genes that specifically target sugar beet pests and pathogens offers a novel means to augment plant breeding to confer disease and insect resistance to an important crop plant. Specific goals of the project are to develop strategies to genetically improve Cercospora leafspot and root maggot resistance based on the manipulation of previously characterized disease resistance genes from heterologous systems and newly discovered sugar beet resistance genes. We have identified and reconstructed a number of beneficial genes for specific expression in sugar beet. We are also developing approaches to identify disease resistance genes in moderately resistant sugar beet varieties. Characterization of the genes will lead to development of novel non-chemical strategies for effective and safe control of sugar beet diseases and pests. This research falls under National Program 302, Plant Biological and Molecular Processes, Component 2B, Understanding Plant Interactions with Their Environment. This project will increase the knowledge and understanding of the basis for resistance to pests and pathogens of a major US crop, sugarbeet. This research will provide 1) knowledge of the regulatory mechanisms that govern plant gene expression; 2) technologies for controlling gene expression to achieve inherent increased productivity and quality characteristics in plant germplasm; and 3) methodology to enhance the development of improved elite germplasm lines with enhanced disease and pest resistance that will lead to increases in yields and the quality and nutritional value of cultivated crops. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2002) 1. Identify known genes with potential for improving Cercospora leafspot resistance in sugar beet, Beta vulgaris L. 2. Develop improved transformation and regeneration protocols for sugar beet, specifically so that transgenic plants can be readily constructed. 3. Determine the feasibility of developing a laboratory bioassay for the sugar beet root maggot using sugar beet seedlings and spinach as a model plant for sugar beet. 4. Initiate the collection of taproot tissues from root maggot infested sugar beet lines (a moderately resistant and a corresponding parental susceptible line) for preparation of cDNA libraries enriched for genes associated with insect and disease resistance. 5. Transform model plants with a reconstructed cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 2 (FY2003) 1. Construct transgenic sugar beet plants carrying the CFP cercosporin transport gene from Cercospora. 2. Verify the genomic insertion of the introduced CFP gene in transgenic sugar beet using PCR with gene-specific primers. 3. Determine the feasibility of developing a root maggot bioassay using sugar beet hairy root cultures derived from moderately resistant and susceptible sugar beet lines for future use as tools for rapid evaluation of newly characterized disease resistance genes. 4. Initiate preparation of cDNA libraries of taproot tissues collected at specific time points following infestation with the root maggot larvae to preferentially select insect and disease resistance genes. 5. Initiate molecular analysis of genetically modified plants transformed with a cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 3 (FY2004) 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. 3. Initiate a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotova. 4. Devise specific primers for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. 5. Introduce a reconstructed reporter gene (GUS) into hairy roots for analysis of tissue-specific and constitutive gene expression in sugar beet root cells. 6. Continue to prepare cDNA libraries from root maggot infested sugar beet tissues for cloning and characterization of disease resistance- associated genes using a subtractive enrichment approach. 7. Molecularly reconstruct proteinase inhibitor genes that specifically target the root maggot digestive proteases for expression in sugar beet hairy root cultures and spinach plants (model for sugar beet) to evaluate the genes' effects on root maggot control. 8. Determine cytochrome P450 protein levels in genetically engineered model plants and relate P450 gene expression to pest and pathogen resistance. Year 4 (FY2005) 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. Begin planting seed from crosses in greenhouses (Beltsville, MD) for evaluation next year. 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. 5. Initiate preparation of sugar beet macroarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures for root maggot resistance analysis using the newly developed root maggot bioassay. 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Year 5 (FY2006) 1. Evaluate Cercospora resistance levels of transgenic plants crossed with agronomically elite germplasms using an artificial field inoculation procedure in the growth chamber. 2. Analysis of genes controlling pathogenesis in the species-specific root rot-causing bacterial phytopathogen (Erwinia betavasculorum) using site direced mutagenesis. 3. Sequence analyses of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. 4. Screen sugar beet macroarrays for genes whose expression is modulated (increased or decreased) by the interaction with the root maggot using molecular probes prepared from sugar beet lines that are moderately resistant and susceptible to the root maggot. 5. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures and spinach plants. 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. Begin planting seed from crosses in greenhouses (Beltsville, MD) for evaluation next year. Milestone Substantially Met 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. Milestone Substantially Met 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. Milestone Substantially Met 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. Milestone Substantially Met 5. Initiate preparation of sugar beet macroarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. Milestone Substantially Met 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures for root maggot resistance analysis using the newly developed root maggot bioassay. Milestone Substantially Met 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. Milestone Fully Met 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The Year 5 milestones are listed below with a description of the anticipated outcomes. The entire project is scheduled to be completed during FY2006 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY2006. Year 5 (FY2006) 1. Evaluate Cercospora resistance levels of transgenic plants crossed with agronomically elite germplasms using an artificial field inoculation procedure in the growth chamber. FY2006: identify particular plant crosses that produced progeny carrying CFP FY2007: identify particular plant crosses that have greater leafspot resaistance FY2008: use the data to plan further crosses 2. Analysis of genes controlling pathogenesis in the species-specific root rot-causing bacterial phytopathogen (Erwinia betavasculorum) using site directed mutagenesis. FY2006: make site-directed mutants in several genes specifying motility FY2007: determine the pathogenicity and relative virulence of these mutants FY2008: examine bacterial mutants for their ability to induce plant defenses 3. Sequence analysis of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. FY2006: shotgun cloning and subcloning of the fragment carrying NPR1 FY2007: assemble and annotate the gene sequence FY2008: identify cis-acting control elements on the BAC clone DNA 4. Screen sugar beet macroarrays for genes whose expression is modulated (increased or decreased) by the interaction with the root maggot using molecular probes prepared from sugar beet lines that are moderately resistant and susceptible to the root maggot. FY2006: identify genes that are associated with either resistance or susceptibility FY2007: characterize genes deemed most valuable for increasing disease and pest resistance; manuscript FY2008: clone full length coding sequences and promoter regions for each gene 5. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures. FY2006: reconstruct genes - fuse the promoter regions to a reporter gene for in planta analysis of temporal and tissue specific expression; fuse the coding sequences to strong promoter for over-expression (sense) or suppression (anti-sense) in planta for analysis of gene function FY2007: generate transformed sugar beet hairy roots carrying the reconstructed genes and promoters for analysis of expression in planta FY2008: identify transformed hairy root cultures that are resistant to the root maggot, other insect pests or microbial pathogens; manuscript 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. FY2006: identify transformed hairy root cultures that are resistant to the root maggot FY2007: molecularly characterized transformed hairy roots; manuscript FY2008: transfer the technology by establishing CRADAs; initiate collaborations with industry 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. FY2006: large numbers of transgenic plants that are resistant to pests and pathogens, will be cultivated for preparation of large quantities of insecticidal extracts for further purification in order to identify the active compound(s) FY2007: extracts analyzed of insecticidal activity; extracts fractionated to identify active fraction FY2008: active compound(s) purified; patent potential evaluated; CRADAs 4a What was the single most significant accomplishment this past year? To improve root maggot and Cercospora resistance in sugarbeet using biotechnological approaches, we needed to identify disease resistance genes and to develop pathogen and insect bioassays for screening newly developed plant varieties for resistance. To characterize sugarbeet resistance genes, we prepared libraries (cDNAs) enriched for root maggot resistance genes from moderately resistant and susceptible breeding lines that were released by ARS scientists in Fargo, ND in collaboration with scientists at North Dakota State University. We identified about 1000 genes half of which were confirmed to be differentially expressed in response to root maggot feeding in the resistant or the susceptible plants. The identification of resistance genes and their introduction into susceptible breeding lines could improve pest and disease resistance, leading to increased sugar yields and reduction in pesticide usage. To improve Cercospora leafspot disease resistance in sugar beet, we generated a transgenic sugar beet plant that expresses a resistance gene derived from a soybean pathogen Cercospora kikuchi. Progeny of the transgenic plant bolted early, negating the possibility of obtaining vegetative plants of sufficient maturity and size for disease evaluation. Therefore, progeny seed were sent to ARS scientists at Ft. Collins, CO for breeding with high quality genotypes C842 and 9933 developed by ARS scientists at Salinas, CA. Seeds harvested from 14 of the 31 crosses are already available and they will be screened for enhanced resistance to the Cercospora pathogen in order to identify resistant germplasm that may be suitable for breeding programs. 4b List other significant accomplishments, if any. In a new genetic study of pathogenesis/virulence in Erwinia betavasculorum, motility and chemotaxis were found to be avirulence factors which are presumed to induce host defense mechanisms and interfere with pathogenesis. Virulent mutants were selected by plant interaction and shown to be impaired in both motility and chemotaxis. We devised specific gene markers (DNA primers) useful for identifying sugar beet genes homologous to the disease resistance-determining gene of Arabidopsis (NPR1) and these have preliminarily been used to identify large stretches of sugarbeet DNA (BAC clones) that may contain the sugar beet NPR1 gene. Characterization of the gene's controlling mechanisms may be useful for increasing disease and pest resistance in sugar beet. Methodology was established for generating sugarbeet root cultures that can be propagated in the laboratory (hairy root cultures). Using hairy roots as a model system permits rapid evaluation of the effects of newly identified resistance genes and toxic compounds on sugarbeet pests and pathogens that specifically target the taproot and rhizosphere. It also provides a means for studying the interaction between sugarbeet roots and the sugarbeet root maggot that may lead to design of novel control approaches that disrupt this interaction. (manuscript in review) We demonstrated that the expression of a defense-related cytochrome P450 gene in transgenic plants enhanced their resistance to insects and pathogenic microbes. Levels of expression of the P450 gene in transgenic plants were determined in order to correlate them with the observed levels of disease resistance. Insecticidal compounds were localized to the leaf surfaces by preparation and analysis of leaf extracts (manuscript published). Collaborative efforts are being explored to purify and identify the active compound(s). Plant transformation vectors were constructed for use as cassettes that allow for rapid cloning of newly discovered resistance genes and regulatory switches for subsequent introduction into plants for analysis of gene function and expression. Sugarbeet were transformed with reconstructed proteinase inhibitor genes that specifically target the sugarbeet root maggot digestive enzymes and regenerated hairy root cultures were regenerated for analysis of resistance. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Identified the CFP gene from Cercospora as having potential for improving Cercospora leafspot resistance in sugar beet and used a newly developed improved transformation and regeneration protocol to construct transgenic plants carrying the CFP gene. We published 2 papers in a peer reviewed journal on the construction of transgenic sugar beet plants carrying the CFP cercosporin transport gene, from Cercospora. A verified transgenic sugar beet carrying the cfp gene has produced viable seed that has already begun to be explored as a potential source of a gene conferring leaf spot resistance. An in vitro sugar beet root maggot bioassay was developed using both sugar beet seedlings and hairy root cultures that will facilitate rapid screening of newly developed sugar beet germplasm for resistance to the root maggot, the most devastating insect pest of sugar beet, and potentially other sugar beet pests and pathogens. (manuscript submitted) We developed improved gene transfer methodology for sugar beet in order to facilitate the introduction of beneficial genes into sugar beet for improved disease and insect resistance. We developed a method to transform embryogenic sugar beet suspension cultures with reporter genes using the biolistic transformation method (manuscript published); we optimized the generation of embryogenic leaf callus from commercially important sugar beet lines for use with the biolistic transformation method and successfully regenerated transgenic plants that express an introduced transgene (manuscripts published). We developed genetically modified plants with a reconstructed gene that is involved in the synthesis of plant compounds that have insecticidal and pharmaceutical properties. Model plants expressing the defense related gene were resistant to insects and microbial pathogens leading to the possibility of utilizing this gene in other plants. (manuscript published) Sugar beet taproot cDNA libraries enriched for defense response genes were prepared and will facilitate the identification and characterization of genes involved in defense mechanisms. Biotechnological strategies to control diseases and pests will be designed based on the modulation of expression of these defense related genes in plants. (proceedings manuscript in press) 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Annual reports to The Beet Sugar Development Foundation and the American Society of Sugar Beet Technologists (ASSBT) have been written and submitted and the BSDF has otherwise been periodically informed of research progress with due diligence to promote scientific advance. Attendance and active participation at the 33rd Biennial ASSBT meeting, ARS sponsored sugar beet workshop and the Beltsville Agricultural Field Day fostered the transfer of biotechnology information to industry and to the consumer. A CRADA was continued for exploring the production of renewable biomass fuel from sugar beet that ultimately would benefit the sugar beet grower and processor industries. A TRUST agreement was continued for identifying disease resistance traits to improve sugar beet for industry and processor groups. An SCA with a university collaborator led to the isolation of plant genes induced by feeding insects that will be developed for use as potential targets for pest control. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Smigocki, A. 2004. Improvement of root maggot and disease resistance in sugar beet. Beet Sugar Development Foundation Res. Rep. E3-10. Wozniak, C.A. and A.C. Smigocki. 2004. Syngliocladium tetanopsis as a fungal biocontrol agent to control the sugarbeet root maggot. Beet Sugar Development Foundation Res. Rep. E13-15.

Impacts
(N/A)

Publications

  • Ivic-Haymes, S.D., Boetel, M., Campbell, L.G., Dregseth, R., Smigocki, A.C. 2005. An in vitro sugar beet root maggot (tetanops myopaeformis) feeding assay. American Society of Sugarbeet Technologists. p.33.
  • Puthoff, D.P., Smigocki, A.C. 2005. Sugar beet genes regulated by sugar beet root maggot (tetanops myopaeformis) infestation. American Society of Sugarbeet Technologists. p.34.
  • Smigocki, A.C., Ivic-Haymes, S., Puthoff, D.P. 2005. Sugar beet root maggot (tetanops myopaeformis) as a model system for elucidating disease and pest resistance mechanisms in roots. American Phytopathological Society Potomac Division Meeting. p.33.
  • Puthoff, D.P., Ivic-Haymes, S., Smigocki, A.C. 2005. Elucidation of resistance and disease mechanisms in roots using sugar beet and sugar beet root maggot as a model system. International Society of Chemical Ecology Meeting. p. 79.
  • Kuykendall, L.D., Young, J., Martinez-Romero, E., Kerr, A., Sawada, H. Rhizobium. 2005. Bergey's Manual of Systematic Bacteriology. pp.325-340.
  • Kuykendall, L.D., Dazzo, F. Allorhizobium. 2005. Bergey's Manual of Systematic Bacteriology. pp. 345, 346.
  • Kuykendall, L.D., Hasmem, F.M., Wang, E., Young, J.M. Sinorhizobium. 2005. Bergey's Manual of Systematic Bacteriology. pp. 358-361.
  • Thomas, J.C., Perron, M., Larosa, P.C., Smigocki, A.C. 2005. Cytokinin and metals regulate a tobacco metallothionein-like gene. Physiologia Plantarum. 123-262:271.
  • Smigocki, A.C., Haymes, S.D., Wilson, D. 2004. Pest and disease resistance enhanced by heterologous suppression of a nicotiana plumbaginifolia cytochrome p450 gene cyp72a2. Biotechnology Letters. 26:1809-1814.
  • Ivic-Hayes, S.D., Smigocki, A.C. 2005. Biolistic transformation of sugar beet (beta vulgaris l.) leaves with high regeneration potential. Plant Cell Reports. 23:699-704.


Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Agronomic losses due to disease - producing bacteria, fungi and insects can be reduced by the genetic construction of transgenic plants carrying genes for the production of antimicrobial and insecticidal proteins and compounds. Environmentally friendly means of controlling disease producing microbes and insects are needed in order to reduce use of chemical pesticides in farming. Some of the major diseases in sugar beet are caused by fungi and bacteria: Aphanomyces, Cercospora, Rhizoctonia, and Erwinia, respectively. Cercospora-induced leafspot and Erwinia- induced root rot are major disease problems affecting the profitability and sustainability of sugar beet production in most areas where the crop is grown. The major insect pest of sugar beet in United States is the root maggot (Tetanops myopaeformis). We are developing and optimizing methods for efficient introduction of beneficial genes into sugar beet. We have identified and reconstructed a number of beneficial genes for their specific expression in sugar beet. We are also developing methodology to identify disease resistance genes in sugar beet germplasm. This non-chemical approach will result in a more effective and safe control of microbial diseases and insect pests in sugar beet. Sugar production from sugar beet has not increased for the last 20 years mainly due to pathogenic microbes and insects. Estimates of the economic losses due to diseases caused by Aphanomyces, Cercospora, Erwinia, and Rhizoctonia microbes vary from 20 to 30% and in some years could even be higher. The sugar beet root maggot has become established at economically damaging levels in at least half of all U.S. acreage and can account for about 23 percent loss in recoverable sugars. Chemical control is becoming less effective due to the increasingly widespread occurrence of resistant pathogens. Some chemical agents are being taken off the market by the EPA. The introduction of reconstructed beneficial genes that specifically target sugar beet pests and pathogens offers a novel means, to augment plant breeding, of conferring disease and insect resistance to important crop plants, with reduced usage of harmful chemical pesticides. Specific goals of the project are to develop strategies to genetically improve Cercospora leafspot and root maggot resistance based on the manipulation of previously characterized disease resistance genes and newly discovered genes. This research falls under National Program 302, Plant Biological and Molecular Processes, and focuses on goals 1 and 3 of the National Program Action Plan. This research approach will fulfill the need for fundamental, long term research to improve sugar beet crop production, protection, product value and safety. Research focuses on improved crop protection to control pests, primarily insects, in a more environmentally friendly manner. This research will provide knowledge of the regulatory mechanisms that govern plant gene expression in order to achieve inherent increased productivity and quality characteristics in plant germplasm. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2002) 1. Identify known genes with potential for improving Cercospora leafspot resistance in sugar beet, Beta vulgaris L. 2. Develop improved transformation and regeneration protocols for sugar beet, specifically so that transgenic plants can be readily constructed. 3. Determine the feasibility of developing a laboratory bioassay for the sugar beet root maggot using sugar beet seedlings and spinach as a model plant for sugar beet. 4. Initiate the collection of taproot tissues from root maggot infested sugar beet lines ( a moderately resistant and a corresponding parental susceptible line) for preparation of cDNA libraries enriched for genes associated with insect and disease resistance. 5. Transform model plants with a reconstructed cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 2 (FY2003) 1. Construct transgenic sugar beet plants carrying the CFP cercosporin transport gene from Cercospora. 2. Verify the genomic insertion of the introduced CFP gene in transgenic sugar beet using PCR with gene-specific primers. 3. Determine the feasibility of developing a root maggot bioassay using sugar beet hairy root cultures derived from moderately resistant and susceptible sugar beet lines for future use as tools for rapid evaluation of newly characterized disease resistance genes. 4. Initiate preparation of cDNA libraries of taproot tissues collected at specific time points following infestation with the root maggot larvae to preferentially select insect and disease resistance genes. 5. Initiate molecular analysis of genetically modified plants transformed with a cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 3 (FY2004) 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. This milestone was not met due to the pitfall described as follows: 3. Initiate a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotova. 4. Devise specific primers for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. 5. Introduce a reconstructed reporter gene (GUS) into hairy roots for analysis of tissue-specific and constitutive gene expression in sugar beet root cells. 6. Continue to prepare cDNA libraries from root maggot infested sugar beet tissues for cloning and characterization of disease resistance- associated genes using a subtractive enrichment approach. 7. Molecularly reconstruct proteinase inhibitor genes that specifically target the root maggot digestive proteases for expression in sugar beet hairy root cultures and spinach plants (model for sugar beet) to evaluate the genes' effects on root maggot control. 8. Determine cytochrome P450 protein levels in genetically engineered model plants and relate P450 gene expression to pest and pathogen resistance. Year 4 (FY2005) 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. 5. Initiate preparation of sugar beet microarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures and spinach for root maggot resistance analysis using the newly developed root maggot bioassay. 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Year 5 (FY2006) 1. Continue crosses of sugar beet lines aimed at developing a biennial CFP transgenic suitable for evaluating Cercospora leafspot resistance. 2. Sequence analysis of genes controlling pathogenesis with sugar beet of the species-specific root rot-causing bacterial phytopathogen (Pectobacterium betavasculorum). 3. Sequence analyses of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. 4. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures and spinach plants. 5. Screen sugar beet microarrays for genes whose expression is modulated (increased or decreased) by the interaction with the root maggot using molecular probes from sugar beet lines that are moderately resistant and susceptible to the root maggot. 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. 3. Milestones: The milestones listed below were scheduled to be completed under Year 3 (FY2004). These milestones were substantially met except as noted. 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. This milestone was not met due to the pitfall described as follows: We successfully produced plants of CFP transgenics and collected seeds that were viable, but the planned evaluation of CFP-carrying transgenics for Cercospora resistance was not done since all of the viable seeds produced only immature plants that bolted early, flowering at the expense of vegetative growth, which resulted in the lack of mature leaves for Cercospora leafspot infection analysis (only mature leaves are susceptible). This pitfall has now been addressed by initiating collaboration with ARS scientists at Ft. Collins CO, who will use the CFP transgenic seeds we sent him to produce plants to cross with an agronomically elite biennial plant so that a biennial transgenic appropriate for leafspot susceptibility evaluation can be derived. This process will likely take at least three years. 3. Initiate a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotovora. 4. Devise specific primers for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. 5. Introduce a reconstructed reporter gene (GUS) into hairy roots for analysis of tissue-specific and constitutive gene expression in sugar beet root cells. 6. Continue to prepare cDNA libraries from root maggot infested sugar beet tissues for cloning and characterization of disease resistance- associated genes using a subtractive enrichment approach. 7. Molecularly reconstruct proteinase inhibitor genes that specifically target the root maggot digestive proteases for expression in sugar beet hairy root cultures and spinach plants (model for sugar beet) to evaluate the genes' effects on root maggot control. 8. Determine cytochrome P450 protein levels in genetically engineered model plants and relate P450 gene expression to pest and pathogen resistance. 1. FY2005, 2006 milestones (new CRIS writeup due FY2006 The Year 4 and 5 milestones are listed below with a description of the anticipated outcomes. The entire project is scheduled to be completed during FY2006 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY2006. Year 4 (FY2005) 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. 5. Initiate preparation of sugar beet microarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures and spinach for root maggot resistance analysis using the newly developed root maggot bioassay. 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Year 5 (FY2006) 1. Continue crosses of sugar beet lines aimed at developing a biennial CFP transgenic suitable for evaluating Cercospora leafspot resistance. 2. Sequence analysis of genes controlling pathogenesis with sugar beet of the species-specific root rot-causing bacterial phytopathogen (Pectobacterium betavasculorum). 3. Sequence analyses of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. 4. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures and spinach plants. 5. Screen sugar beet microarrays for genes whose expression is modulated (increased or decreased) by the interaction with the root maggot using molecular probes from sugar beet lines that are moderately resistant and susceptible to the root maggot. 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 (one per Research Project): In order to improve the Cercoosora leafspot and root maggot resistance in sugar beet using biotechnological approaches, we needed to identify disease resistance genes and to develop pathogen and insect bioassays for screening newly developed plant varieties for resistance. The expression of a cercosporin toxin export gene in transgenic sugar beets was determined by transcription (RT-PCR) and translation (Western blot) analyses and published in a peer-reviewed journal; scientists from the Molecular Plant Pathology Laboratory at Beltsville, Md in cooperation with scientists from North Dakota State University developed transformed sugar beet root cultures (hairy roots) for rapid evaluation of resistance genes in plant cells; and Beltsville scientists demonstrated insect and bacterial resistance in transgenic model plants that was mediated by the expression of a cytochrome P450 gene involved in the synthesis of secondary metabolites. Root maggot damaged tissues from susceptible and relatively resistant sugar beet varieties released by ARS scientists in Fargo, ND were collected for characterization of insect resistance genes. The identification of resistance genes and their introduction into susceptible plants could improve disease resistance, leading to increased sugar yields and reduction in fungicide and pesticide usage. B. Other Significant Accomplishment (s), if any. We initiated a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotovora. This new species is relatively uncharacterized and it is specific for sugar beets. We devised specific primers useful for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. The plan is to identify BAC clones from a sugar beet genetic library carrying NPR1 genes and to sequence the DNA. To improve disease resistance in agronomically important sugar beet breeding lines using biotechnological approaches, we needed to develop improved methodology to enhance the regeneration and transformation of sugar beet. We developed a method to transform embryogenic sugar beet suspension cultures with reporter genes using the biolistic transformation method (manuscript published); we optimized the generation of embryogenic leaf callus from commercially important sugar beet lines for use with the biolistic transformation method and successfully regenerated transgenic plants that express an introduced transgene (manuscript in press). The improved transformation methodology will facilitate the introduction of beneficial genes to sugar beet. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Agronomic losses due to disease - producing bacteria, fungi and insects can be reduced by the genetic construction of transgenic plants carrying genes for the production of antimicrobial and insecticidal proteins and compounds. Environmentally friendly means of controlling disease producing microbes and insects are needed in order to reduce use of chemical pesticides in farming. Some of the major diseases in sugar beet are caused by fungi and bacteria: Aphanomyces, Cercospora, Rhizoctonia, and Erwinia, respectively. Cercospora-induced leafspot and Erwinia- induced root rot are major disease problems affecting the profitability and sustainability of sugar beet production in most areas where the crop is grown. The major insect pest of sugar beet in United States is the root maggot (Tetanops myopaeformis). We are developing and optimizing methods for efficient introduction of beneficial genes into sugar beet. We have identified and reconstructed a number of beneficial genes for their specific expression in sugar beet. We are also developing methodology to identify disease resistance genes in sugar beet germplasm. This non-chemical approach will result in a more effective and safe control of microbial diseases and insect pests in sugar beet. Sugar production from sugar beet has not increased for the last 20 years mainly due to pathogenic microbes and insects. Estimates of the economic losses due to diseases caused by Aphanomyces, Cercospora, Erwinia, and Rhizoctonia microbes vary from 20 to 30% and in some years could even be higher. The sugar beet root maggot has become established at economically damaging levels in at least half of all U.S. acreage and can account for about 23 percent loss in recoverable sugars. Chemical control is becoming less effective due to the increasingly widespread occurrence of resistant pathogens. Some chemical agents are being taken off the market by the EPA. The introduction of reconstructed beneficial genes that specifically target sugar beet pests and pathogens offers a novel means, to augment plant breeding, of conferring disease and insect resistance to important crop plants, with reduced usage of harmful chemical pesticides. Specific goals of the project are to develop strategies to genetically improve Cercospora leafspot and root maggot resistance based on the manipulation of previously characterized disease resistance genes and newly discovered genes. This research falls under National Program 302, Plant Biological and Molecular Processes, and focuses on goals 1 and 3 of the National Program Action Plan. This research approach will fulfill the need for fundamental, long term research to improve sugar beet crop production, protection, product value and safety. Research focuses on improved crop protection to control pests, primarily insects, in a more environmentally friendly manner. This research will provide knowledge of the regulatory mechanisms that govern plant gene expression in order to achieve inherent increased productivity and quality characteristics in plant germplasm. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2002) 1. Identify known genes with potential for improving Cercospora leafspot resistance in sugar beet, Beta vulgaris L. 2. Develop improved transformation and regeneration protocols for sugar beet, specifically so that transgenic plants can be readily constructed. 3. Determine the feasibility of developing a laboratory bioassay for the sugar beet root maggot using sugar beet seedlings and spinach as a model plant for sugar beet. 4. Initiate the collection of taproot tissues from root maggot infested sugar beet lines (a moderately resistant and a corresponding parental susceptible line) for preparation of cDNA libraries enriched for genes associated with insect and disease resistance. 5. Transform model plants with a reconstructed cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 2 (FY2003) 1. Construct transgenic sugar beet plants carrying the CFP cercosporin transport gene from Cercospora. 2. Verify the genomic insertion of the introduced CFP gene in transgenic sugar beet using PCR with gene-specific primers. 3. Determine the feasibility of developing a root maggot bioassay using sugar beet hairy root cultures derived from moderately resistant and susceptible sugar beet lines for future use as tools for rapid evaluation of newly characterized disease resistance genes. 4. Initiate preparation of cDNA libraries of taproot tissues collected at specific time points following infestation with the root maggot larvae to preferentially select insect and disease resistance genes. 5. Initiate molecular analysis of genetically modified plants transformed with a cytochrome P450 gene that is associated with the production of insecticidal secondary metabolites in plants. Year 3 (FY2004) 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. This milestone was not met due to the pitfall described as follows: 3. Initiate a new genetic study of pathogenesis/virulence in Pectobacterium betavasculorum, formally a subspecies of Erwinia carotova. 4. Devise specific primers for identifying sugar beet genes homologous to the disease resistance-determining NPR1 gene of Arabidopsis. 5. Introduce a reconstructed reporter gene (GUS) into hairy roots for analysis of tissue-specific and constitutive gene expression in sugar beet root cells. 6. Continue to prepare cDNA libraries from root maggot infested sugar beet tissues for cloning and characterization of disease resistance- associated genes using a subtractive enrichment approach. 7. Molecularly reconstruct proteinase inhibitor genes that specifically target the root maggot digestive proteases for expression in sugar beet hairy root cultures and spinach plants (model for sugar beet) to evaluate the genes' effects on root maggot control. 8. Determine cytochrome P450 protein levels in genetically engineered model plants and relate P450 gene expression to pest and pathogen resistance. Year 4 (FY2005) 1. Perform plant crosses with CFP transgenics and agronomically elite biennial germplasm to obtain plants desirable for evaluation of Cercospora leafspot resistance, in cooperation with ARS scientists at Ft. Collins, CO. 2. Identify genes for virulence or avirulence in Pectobacterium betavasculorum. 3. Screen a BAC genomic library of sugar beet for genes homologous to the NPR1 gene of Arabidopsis in cooperation with ARS scientists at East Lansing, MI. 4. Finish preparing and screening cDNA libraries from root maggot infested sugar beet tissues and characterize putative disease resistance genes by nucleotide sequence determination and blast search analyses. 5. Initiate preparation of sugar beet microarrays of gene clones isolated from cDNA libraries to identify a suite of genes whose expression is modulated by the interaction with the root maggot. 6. Introduce reconstructed proteinase inhibitor and cytochrome P450 genes into sugar beet hairy root cultures and spinach for root maggot resistance analysis using the newly developed root maggot bioassay. 7. Evaluate transgenic plants carrying the reconstructed cytochrome P450 gene for resistance to bacteria, fungi and insects. 8. Establish collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Year 5 (FY2006) 1. Continue crosses of sugar beet lines aimed at developing a biennial CFP transgenic suitable for evaluating Cercospora leafspot resistance. 2. Sequence analysis of genes controlling pathogenesis with sugar beet of the species-specific root rot-causing bacterial phytopathogen (Pectobacterium betavasculorum). 3. Sequence analyses of bacterial artificial chromosomes that carry genes homologous to the NPR1 gene to identify controlling elements that may be useful to enhance the expression of multiple disease resistance pathways. 4. Reconstruct putative disease resistance genes isolated from the sugar beet cDNA library screens for structural and functional analyses in sugar beet hairy root cultures and spinach plants. 5. Screen sugar beet microarrays for genes whose expression is modulated (increased or decreased) by the interaction with the root maggot using molecular probes from sugar beet lines that are moderately resistant and susceptible to the root maggot. 6. Evaluate sugar beet hairy root cultures transformed with reconstructed proteinase inhibitor, cytochrome P450 and newly identified resistance genes for resistance to the root maggot. 7. Continue collaborations to identify the secondary metabolites associated with disease resistance in the transgenic cytochrome P450 plants. Question 3. The milestones listed below were scheduled to be completed under Year 3 (FY2004). These milestones were substantially met except as noted. 1. Determine the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). 2. Grow mature plants of CFP transgenics and collect seeds that are viable. This milestone was not met due to the pitfall described as follows: We successfully produced plants of CFP transgenics and collected seeds that were viable, but the planned evaluation of CFP-carrying transgenics for Cercospora resistance was not done since all of the viable seeds produced only immature plants that bolted early, flowering at the expense of vegetative growth, which resulted in the lack of mature leaves for Cercospora leafspot infection analysis (only mature leaves are susceptible). This pitfall has now been addressed by initiating . Identified the CFP gene from Cercospora as having potential for improving Cercospora leafspot resistance in sugar beet and used a newly developed improved transformation and regeneration protocol to construct transgenic plants carrying the CFP gene. We published a paper in a peer reviewed journal on the construction of transgenic sugar beet plants carrying the CFP cercosporin transport gene, from Cercospora, and their verification as carrying the CFP gene using PCR with gene-specific primers. We published a peer-reviewed journal article on the expression of CFP in transgenic sugar beets both in terms of transcription (RT-PCR) and translation (Western blot). A putative transgenic sugar beet carrying the cfp gene has been produced as a potential source of new germplasm for leaf spot resistance. An in vitro sugar beet root maggot bioassay was developed using both sugar beet seedlings and hairy root cultures that will facilitate 1) the screening of newly developed sugar beet germplasm for resistance to the root maggot, the most devastating insect pest of sugar beet, and 2) the collection of infested tissues for molecular analysis intended to identify and characterize a suite of genes associated with disease resistance in sugar beet. We developed improved gene transfer methodology for sugar beet in order to facilitate the introduction of beneficial genes into sugar beet for improved disease and insect resistance and published the results in peer- reviewed journals. We developed transgenic model plants with a reconstructed gene that is involved in the synthesis of plant compounds that have insecticidal and pharmaceutical properties. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The Beet Sugar Development Foundation and the American Association of Sugar Beet Technologists have both been periodically informed of research progress with due diligence to foster scientific advance. Attendance and active participation as an invited speaker and sugar beet workshop participant at national and international meetings as well as participation in the Beltsville Agricultural Field Day transferred biotechnology information to industry and to the consumer, respectively. A CRADA was continued for exploring the production of renewable biomass fuel from sugar beet that ultimately would benefit the sugar beet grower and processor industries. A TRUST agreement was continued for identifying disease resistance traits to improve sugar beet for industry and processor groups. An SCA was established to characterize insect- induced genes in plants as potential targets for pest control. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Smith, G. June 2004. Biomethanol from sugar beet pulp. Sugar Journal, p. 11.

Impacts
(N/A)

Publications

  • Kuykendall, L.D., Upchurch, R.G. 2004. Expression in sugar beet of the introduced cercosporin toxin export gene cfp from cercospora kikuchii, the causative organism of purple seed stain in soybean. Biotechnology Letters. 26:723-727.
  • Smigocki, A.C., Campbell, L.G., Wozniak, C.A. 2003. Leaf extracts from cytokinin-overproducing transgenic plants kill sugarbeet root maggot (tetanops myopaeformis) larvae. Journal of Sugarbeet Research. 40(4):1-11.
  • Ivic, S.D., Smigocki, A.C. 2003. Transformation of sugar beet cell suspension cultures. In Vitro Cellular And Developmental Biology. 39(6):1- 6.
  • Smigocki, A.C. 2004. Multifaceted molecular approach for control of the sugar beet root maggot (tetanops myopaeformis roder). Plant and Animal Genome Abstracts. page 68.
  • Smigocki, A.C. 2003. Beneficial genes for control of the sugar beet root maggot (tetanops myopaeformis roder). Entomological Society of America Annual Meeting. p. 231.


Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Agronomic losses due to disease-producing bacteria, fungi and insects can be reduced by the genetic construction of transgenic plants carrying genes for the production of antimicrobial and insecticidal proteins and compounds. Environmentally friendly means of controlling disease- producing microbes and insects are needed in order to reduce use of chemical pesticides in farming. Some of the major diseases in sugar beet are caused by fungi and bacteria: Aphanomyces, Cercospora, Rhizoctonia, and Erwinia, respectively. The major insect pest of sugar beet in United States is the root maggot (Tetanops myopaeformis). We are developing and optimizing methods for efficient introduction of beneficial genes into sugar beet. We have identified and reconstructed a number of beneficial genes for their specific expression in sugar beet. We are also developing methodology to identify disease resistance genes in sugar beet germplasm. This non-chemical approach will result in a more effective and safe control of microbial diseases and insect pests in sugar beet. 2. How serious is the problem? Why does it matter? Sugar production from sugar beet has not increased for the last 20 years mainly due to pathogenic microbes and insects. Estimates of the economic losses due to diseases caused by Aphanomyces, Cercospora, Erwinia, and Rhizoctonia microbes vary from 20 to 30% and in some years could even be higher. The sugar beet root maggot has become established at economically damaging levels in at least half of all U.S. acreage and can account for about 23 percent loss in recoverable sugars. Chemical control is becoming less effective due to the increasingly widespread occurrence of resistant pathogens. Some chemical agents are being taken off the market by the EPA. The introduction of reconstructed beneficial genes that specially target sugar beet pests and pathogens offers a novel means, to augment plant breeding, of conferring disease and insect resistance to important crop plants, with reduced usage of harmful chemical pesticides. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 302, Plant Biological and Molecular Processes (100%). This research approach will fulfill the need for fundamental, long term research to improve sugar beet crop production, protection, product value and safety. Research focuses on improved crop protection to control pests, primarily insects, in a more environmentally friendly manner. This research will provide knowledge of the regulatory mechanisms that govern plant gene expression in order to achieve inherent increased productivity and quality characteristics in plant germplasm. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 (one per Research Project): In order to improve the Cercoosora leafspot and root maggot resistance in sugar beet using biotechnological approaches, we needed to identify disease resistance genes and to develop pathogen and insect bioassays for screening newly developed plant varieties for resistance. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD in cooperation with scientists from North Dakota State University developed a sugar beet root maggot feeding bioassay and determined that the maggot larvae are sensitive to insecticidal compounds from plants genetically engineered to overproduce a growth hormone. By demonstrating that only newly hatched larvae would feed on roots of young seedlings, root maggot damaged tissues from susceptible and relatively resistant sugar beet varieties released by ARS scientists in Fargo, ND were collected for characterization of insect resistance genes. The identification of resistance genes and their introduction into susceptible plants could improve disease resistance, leading to increased sugar yields and reduction in fungicide and pesticide usage. B. Other Significant Accomplishment (s), if any. Our goal was to determine whether the cfp gene coding for transport or export of cercosporin toxin can increase resistance to leaf spot disease in sugar beet. A transgenic plant was vegetatively propagated by scientists of the Molecular Plant Pathology Laboratory at Beltsville, MD. Seeds of the cfp transgenic plant, needed for the development of a new germplasm line, are being produced. The successful development of a leaf spot resistant sugar beet could increase profitability of commercial sugar-beet production by as much as 25%. To determine if genes involved in the production of insecticidal or pharmaceutical compounds can be manipulated in plants to enhance insect resistance or production of value added compounds, we introduced a cytochrome P450 gene into model plants, tobacco and tomato. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD selected three generations of plants with homozygous expression of the P450 gene or plants in which the function of the gene was suppressed. A preliminary screen of the engineered plants indicated that overproduction of the P450 gene product correlates with enhanced insect resistance. Further analysis of the role of the P450 gene in insect resistance could lead to improved disease resistant plant varieties that require reduced levels of harmful chemicals for pest and disease control. For years we have been trying to enhance regeneration and transformation in sugar beet to improve the methodology for molecular genetic manipulation. AFLP analysis was performed by scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD in collaboration with ARS scientists at Colorado State University to investigate the possibility that regenerated sugar beet clones are different genetically from parental germplasm because of selection. In vitro regenerated clones were found to have the same AFLP pattern as parental lines. This finding eliminates the possibility that regenerated lines could be used as a source of highly regeneratable germplasm and is of interest to other researchers interested in germplasm improvement of sugar beet. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A gene transfer method for introducing foreign genes into sugarbeets was developed and optimized. Antimicrobial (cecropin, osmotin, PRS, thionin, cercospora toxin export protein) and insecticidal (cytokinin biosynthesis gene, proteinase inhibitors) genes targeting major sugar beet pathogens and pests have been identified and reconstructed for expression in sugar beet plants. A transgenic sugar beet carrying the cfp gene from Cercospora has been produced as a potential source of new germplasm for leaf spot resistance. An in vitro sugar beet root maggot bioassay was developed that will facilitate the screening of newly developed sugar beet germplasm for resistance to the root maggot, the most devastating insect pest of sugar beet. 6. What do you expect to accomplish, year by year, over the next 3 years? In FY 2004 - continue analysis of P450 transgenic plants; analyze the physiological role of the P450 gene in insect and disease resistance and in the production of secondary metabolites with pharmaceutical properties; determine the secondary metabolite profiles of plants engineered with the P450 gene; explore the potential use of transgenic plants for molecular farming for biobased products; identify new genes that target major pathogens and pests to improve disease resistance in sugar beet; prepare subtracted cDNA libraries enriched for resistance genes using mRNA prepared from root maggot infested tissues of resistant and susceptible sugar beet lines; begin isolating and characterizing the sugar beet resistance genes; evaluate the cfp transgenics for in vitro cercosporin toxin resistance, multiply the transgenics, and evaluate Cercospora leaf spot susceptibility relative to the parent germplasm. In FY 2005 - isolate and characterize the sugar beet resistance genes in order to devise strategies for improved disease resistance in plants; identify insecticidal secondary metabolites and their corresponding biosynthetic genes; analysis of Cercospora resistance will be completed and seed will be supplied to breeders for further evaluation. In FY 2006 - genes involved in the biosynthesis of insecticidal compounds and resistance genes cloned from sugar beet libraries will be reengineered for tissue specific expression in transgenic plants and introduced into sugar beet and model crops, such as spinach and tomato, to determine their effect on disease resistance. The npr1 gene controlling pest and pathogen resistance in Arabidopsis will be introduced into sugar beet and spinach to determine whether this regulatory gene will enhance disease and pest resistance. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Our attendance and active participation in the 2003 meeting of the American Society of Sugar Beet Technologist/ International Institute for Research on Beets and the Beltsville Agricultural Field Day transferred biotechnology information to industry and to the consumer, respectively. A CRADA was established for exploring the production of renewable biomass fuel from sugar beet that ultimately would benefit the sugar beet grower and processor industries. A TRUST was established for identifying disease resistance traits to improve sugar beet for industry and processor groups. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Washington Post, Monday 23 December 2002, MD TEDCO funds a joint venture between a local company and USDA to explore renewable biofuel production from sugar beet. Frederick Business Week of the Frederick News-Post, Monday 23 December 2002, MD TEDCO funds a joint venture between a local company and USDA to explore renewable biofuel production from sugar beet.

Impacts
(N/A)

Publications

  • Kuykendall, L. D., Stockett, T. M. Transforamtion of sugar beet with cfp to improve Cercospora leafspot resistance. Proceedings of the First Joint International Institute for Beet Research and American Society of Sugar Beet Technologists. 2003. p. 883-886.
  • Kuykendall, L. D., Stockett, T. M. Improving resistance to Cercospora- induced leaf spot disease in sugar beet using biotechnology. Beet Sugar Development Foundation Annual Report. Denver, CO. 2003. p. E14-E17.
  • Kuykendall, L.D., T. M. Stockett, J.W. Saunders. Rhizobium radiobacter conjugation and callus-independent shoot regeneration used to introduce the cercosporin export gene cfp from Cercospora into sugar beet (Beta vulgaris L.). Biotechnology Letters 2003. v. 25. p. 739-744.
  • Samac, D.A., Smigocki, A.C. Expression of oryzacystatin I and II in alfalfa increases resistance to the root-lesion nematode. Phytopathology Journal, 203. v. 7. p. 700-804.
  • Smigocki, A.C., Hue, S., Buta, G. Cytokinins as bioregulators promote insect resistance in plants transformed with the ipt gene. Proceedings of the Plant Growth Regulation Society of America. 2002. p. 2-7.
  • Smigocki, A.C., Ivic, S.D., Wilson, D., Wozniak, C.A., Campbell, L., Dregseth, R., Boetel, M. Molecular approaches for control of the sugar beet root maggot. Proceedings of the First Joint International Institute for Beet Research and American Society of Sugar Beet Technologist. 2003. p. 419-428.
  • Ivic, S.D., Smigocki, A.C. Direct gene transfer to sugar beet leaves. Proceedings of the First Joint International Institute for Beet Research and American Society of Sugar Beet Technologists. 2003. p. 903-908.
  • Smigocki, A.C. Improvement of root maggot resistance in sugar beet with proteinase inhibitor genes. Beet Sugar Development Foundation Annual Report. Denver, CO. 2003. p. E9-E13.
  • Smigocki, A.C. Targeted gene transfer to sugar beet taproots to optimize sucrose yields. Beet Sugar Development Foundation Annual Report. Denver, CO. 2003. p. E2-E8.
  • Smigocki, A.C. Cytokinin induced insect tolerance and regulated expression of a cytochrome P450 gene. Proceedings of the 7th International Congress of Plant Molecular Biology. 2003. p. 133.