Location: Molecular Plant Pathology Laboratory2011 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, 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.
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, 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
Insect damage reduces sugar yields from sugar beet. Biotechnology is being used to make sugar beet more resistant to insect attack and increase sugar yields. Regulatory switches (plant promoters) are needed for steering the production of insecticidal compounds to insect damage sites. We identified several sugar beet switches that direct insect-fighting compounds to the root skin, the whole root or leaves. This information will be used to develop safer approaches of insect control to increase yields and reduce usage of chemical pesticides. To feed the rising global population will require that crops have an increased capacity to utilize shrinking natural resources and to resist diseases and insect pests. We studied plant responses to environmental stresses, primarily high soil salt concentrations due to reduced water availability. Our results demonstrate that a growth regulator gene known to enhance insect resistance supported plant growth in high salt concentrations, extended the growing period and increased yields. This information will be used by scientists to develop crops that are capable of mitigating the effects of environmental stresses. A gene cloned from rice to test for insect resistance in sugar beets was also tested against another serious plant insect. Potato is highly susceptible to attack by the Colorado potato beetle (CPB). To improve resistance, several potato varieties were modified with the beneficial insect resistance trait isolated from rice. When CPB larvae were fed potato leaves modified with the rice resistance trait, larvae did not develop normally and their growth was stunted. This information will be used to develop new varieties of potato that are more resistant to CPB. Using molecular means to improve disease resistance in crop plants requires knowledge of gene content and function within chromosomes. This study found tight physical linkage of three key genes essential for crop productivity and disease resistance. Responsible for walling off microbial invaders and metering light availability, genes “BvCS1 (callose synthase)” and “PhyA1 (phytochrome A)” are near “MAP3Ka” a gene whose product initiates disease resistance. Geneticists can use this information to enhance gene expression. Molecular defense mechanisms in sugar beet were investigated to improve disease resistance. “Callose synthase,” involved in defense, causes intruders to be confined. BvCS1 gene structure was studied, and a new mobile element, “Bert,” lies in an unused area where it doesn’t interfere with expression. Since only nonintrusive Bert elements are found, plant survival seems to require key genes and their manipulation could provide new means for crop improvement. Crop productivity could be approximately doubled if disease and stress problems were resolved. Increased knowledge of genes is needed to control disease and stress resistance. In sugar beet, grape, tomato and poplar, a “Heat Shock Factor-like” developmental gene is immediately adjacent to a gene that controls disease resistance. Scientists could use this knowledge to devise innovative new and effective strategies to improve both disease and stress resistance.
1. Sugar beet genes associated with root defense response mechanisms. Plant diseases and pest problems are responsible for decreases in crop yields. To improve disease resistance, a better understanding of the molecular mechanisms controlling plant defense responses is needed. An enriched technique was utilized to discover and characterize sugar beet root genes involved in disease resistance responses. Sugar beet and model plants were modified with the newly discovered sugar beet resistance genes that were re-designed for high levels of production in plants. This was a first demonstration of genetic modification to enhance insect resistance in sugar beet. Scientists will use this information to identify plant resistance mechanisms that will lead to new methods for developing improved plant varieties with enhanced disease and pest resistance that will increase yields and the quality and nutritional value of cultivated crops.
2. Novel molecular strategies for management of the sugar beet root maggot. 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 sugar yield valued at more than $1.2 billion. A comprehensive approach to target the root maggot included pioneering studies that determined the insect’s digestive enzymes and a novel strategy that characterized both sugar beet and root maggot genes important in their mutual interaction. The discovery of the root maggot midgut digestion process opened novel avenues for developing new root maggot control measures. A unique sugar beet gene that targets the root maggot digestive enzymes was characterized and demonstrated to improve insect resistance in genetically modified plants. A laboratory test for root maggot feeding was developed and provided essential technology for studying the interactions between the host plant and the insect pest. An enrichment technique was adapted for the discovery of root maggot genes important in the interaction of the insect with resistant and susceptible sugar beet roots. Scientists will use this information for rapid screening of germplasm for root maggot resistance and for functional analysis of resistance genes.
3. Knowledge of proximity of genes, called physical “linkage”, is critical information for precise genetic manipulation. Our discovery that certain key core genes exhibit close physical linkage highly conserved over a broad range of crop species is consistent with these genes being important and suggests coordinated expression is under selection. Coordinated responses of key genes is selected due to disease pressure and selection for maximum yield.
Kuykendall, L.D., Burdekin, K.A., Shao, J.Y., Conway, L.B. 2011. A plant gene encodes a 'HSFA9-like' heat shock factor and is part of a cluster of orthologous genes including NPR1, CaMP and CK1 in Beta vulgaris, Populus trichocarpa, Solanum lycopersicum and Vitis vinifera. Advanced Studies in Biology. 3(1):13-24.