Location: Molecular Plant Pathology Laboratory
Project Number: 1245-21220-230-00-D
Project Type: In-House Appropriated
Start Date: Oct 1, 2010
End Date: Jul 22, 2013
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, 3a) 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 3b) evaluate recent disease and insect control approaches that are mediated by genes with demonstrated roles in plant defense mechanisms.
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.