Start Date: Oct 01, 2010
End Date: Jul 22, 2013
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.