2007 Annual Report 1a.Objectives (from AD-416) The overall goal of this project is to gain an understanding of sugar beet defense responses in order to devise biotechnological approaches for more effective disease and pest control to improve yields and sugar production. Specific objectives are to. 1)discover and characterize plant genes important in resistant or susceptible responses of sugar beet to sugar beet root maggot,. 2)identify and characterize pathogen genes and molecular signals responsible for virulence in interactions of sugar beet with Erwinia betavasculorum and determine the potential of avirulent Erwinia mutants to elicit expression of sugar beet defense genes characterized in Objective 1,. 3)design and evaluate new approaches for increasing sugar beet disease and insect resistance by manipulating the expression of genes identified in Objective 1 and 2 as being important in sugar beet root maggot and/or Erwinia interactions with sugar beet roots, and. 4)evaluate recent disease and insect control approaches that are mediated by genes with demonstrated roles in plant defense mechanisms. 1b.Approach (from AD-416) The defense response of sugar beet roots to the sugar beet root maggot (SBRM) is being characterized using suppressive subtractive hybridization of messages induced or suppressed after SBRM infestation in both moderately resistant and susceptible germplasm. Genes that are either up- or down-regulated in the resistant and/or the susceptible germplasm will be identified. Molecular techniques will be used to characterize the structure and function of the cloned genes. Clone characterization will include confirmation of differential expression, sequencing of selected clones, functional grouping of genes based on their sequences, full length cDNA cloning of genes identified as potentially having a role in resistance, and expression profiling following various plant stresses that include mechanical wounding, pathogen infection and other well-recognized defense response elicitors. Selected genes will be reconstructed for plant expression or suppression in sugar beet hairy root cultures for analysis of resistance to the sugar beet root maggot or Erwinia pathogen. Targeting expression to the site of insect or pathogen attack will be achieved by reconstructing resistance genes with taproot-specific promoters of genes we identify as being highly expressed in roots. Heterologous and homologous manipulation of the NPR1 gene of Arabidopsis will be followed since NPR1 is a centrally important regulatory gene that controls several different pathways of induced defense responses to microbial pathogens and insect pests. We will compare the NPR1-controlling sequences in both susceptible and resistant genotypes, as for example, C60 and HS11 in the case of Erwinia. We will make site-directed mutants of E. betavasculorum in the genes homologous to the hexA, hexY and in fla, fli and flm genes involved in flagellin synthesis since the hexA and hexY genes of E. carotovora have been implicated in controlling both virulence and motility. E. betavasculorum mutants will be evaluated for pathogenicity and virulence on sugar beet. We will express proteinase inhibitor (PI) transgenes in sugar beet hairy root cultures that we demonstrated specifically inhibit SBRM digestive proteases. We will bioassay transgenic sugar beet that express the reconstructed PI genes for resistance to SBRM and other insect pests that utilize similar mechanistic classes of digestive proteases for assimilation of nutrients from consumed food. We will pyramid inhibitor genes to enhance the stability of the PIs in the insect midguts and to inhibit the activity of digestive proteases not targeted with single PIs as a strategy to enhance plant resistance to insects. 3.Progress Report The NPR1 gene in Arabidopsis is centrally important in enabling the plant to effectively resist microbial and pest attack. To clone the sugar beet NPR1 gene, about 34,500 BAC clones carrying randomly-generated, overlapping DNA fragments representing the entire sugar beet genome were examined and one clone was determined to carry the NPR1 gene and adjacent DNA sequences. A promoter-like region was identified and compared with that of Arabidopsis thaliana. The characterization of over 450 sugar beet root ESTs (GenBank accessions DV501516 - DV501974) associated with root defense response mechanisms was continued. Macroarray technology was used to decipher the regulation of the ESTs by wounding and defense response elicitors (salicylic acid, jasmonic acid, ethylene). Molecular characterization of one of the cloned root genes whose sequence revealed a potential function in root maggot resistance has been initiated. The full length coding sequence of the gene was cloned and reconstructed for over-expression in plant cells. Sugar beet was transformed to generate transformed hairy root tissues as a model root system for analysis of this gene's function in disease resistance mechanisms. Similarly, the gene promoter was cloned, fused to a reporter gene and introduced into sugar beet for in planta analysis of gene expression patterns incited by biotic and abiotic stresses. Unfortunately, almost all of these lines were lost when a growth chamber overheated and a monitoring system failed. The few remaining lines are being molecularly analyzed for foreign gene expression levels and insect and disease resistance. The molecular analysis of transformed sugar beet hairy root cultures that express proteinase inhibitor (PI) genes that target insect digestive enzymes was continued. Over 250 independently derived hairy root lines of both root maggot susceptible and moderately resistant germplasm were engineered to express 9 different PI gene constructs expressed from various promoters. Unfortunately, almost all of these lines were lost when a growth chamber overheated and a monitoring system failed. The few remaining lines are being molecularly analyzed for foreign gene expression levels and insect resistance. Model plants (Nicotiana benthamiana) were transformed with the PI gene constructs for analysis of resistance to leaf feeding insects and microbial pathogens. As with the sugar beet hairy roots, almost all of the transgenic plants were lost when a growth chamber overheated and a monitoring system failed. The few remaining transgenic plants are being molecularly analyzed for foreign gene expression levels and insect resistance. Analysis of more than 20 independently derived transgenic plants that express 6 different defense-related cytochrome P450 gene constructs was completed. Temporal and tissue specific pattern of transgene expression was determined and related to the observed increased level of disease and insect resistance. Extracts prepared from resistant plants were screened for insecticidal activity and selected for further purification of the insecticidal compound(s). 4.Accomplishments Documented major overall similarity in structure and established some minor differences exhibited by NPR1 that is critically important to effective disease resistance in sugar beet and in Arabidopsis. Consistent with ARS National Program 302: Plant Biological and Molecular Processes, Component I Analysis and Modification of Plant Genomes: molecular characterization of plant genes (DNA sequence analysis and gene annotation). A knowledge gap concerning genetic elements controlling disease resistance mechanisms in crop plants and in model plants such as Arabidopsis limits the ability of scientists to use genomics information to improve disease resistance in crop plants. In this project, a promoter-like positive control region just upstream of the sugar beet NPR1 disease resistance control gene was identified and its molecular structure was compared with that of Arabidopsis thaliana. An important step in molecular genetic analysis aimed at improving expression of the sugar beet NPR1 to enhance overall disease resistance, knowledge of adjacent genetic sequences modulating expression of a key pathogen resistance-controlling gene in plants will help plant molecular geneticists devise strategies to enhance disease and pest resistance in agriculturally important crops. Disease and pest problems are responsible for decreases in yield of sugar from sugar beet. The sugar beet root maggot is one of the most devastating insect pests of sugar beet that is found in two-thirds of all U.S. sugar beet fields and accounts for 10-100% reduction in yields. Ineffective cultural practices and harmful chemical insecticides are the only available measures for control of the root maggot; therefore, a strong impetus exists for development of effective and environmentally friendly control measures. We identified sugar beet genes that are associated with root responses to root maggot feeding in both susceptible and moderately resistant sugar beet varieties. Information on the root responses will increase our knowledge of plant-insect interactions. Scientists will use this information to identify plant resistance mechanisms that will lead to new approaches for increasing root maggot resistance in economically important sugar beet varieties without the use of harmful pesticides. Accomplishment contributes to NP 302, Component 2B by increasing knowledge and understanding of the basis for resistance to pests and pathogens of a major U.S. crop, sugar beet. 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. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of new CRADAs and MTAs 1 Number of active CRADAs and MTAs 3 Number of non-peer reviewed presentations and proceedings 8 Number of newspaper articles and other presentations for non-science audiences 4 Review Publications Kuykendall, L.D., Murphy, T.S., Shao, J.Y., Mcgrath, J.M. 2007. Nucleotide Sequence Analyses of a Sugar Beet Genomic NPR1-class Disease Resistance Gene. Journal of Sugarbeet Research. 44:35-49. Smigocki, A.C., Puthoff, D.P., Ivic-Haymes, S.D., Zuzga, S. 2007. A beta vulgaris serine proteinase inhibitor gene (bvsti) regulated by sugar beet root maggot feeding on moderately resistant f1016 roots. American Society of Sugarbeet Technologists Proceedings. 34:143-150. Uzelac, B., Ninkovic, S., Smigocki, A.C., Budimir, S. 2007. Origin and development of secondary somatic embryos in transformed embryogenic cultures of medicago sativa l. cv. zajecarska 83. Biologia Plantarum. 51(1):1-6.