Location: Molecular Plant Pathology Laboratory2015 Annual Report
1a. Objectives (from AD-416):
The goal of this project is to devise, develop, and implement novel, advanced biotechnologies to improve pest and disease resistance in sugar beet that will lead to an improvement in yield and sugar production. Molecular understanding of how plants protect themselves from insect attack and the complementary insect responses to interactions with host plants will be deciphered. Specific objectives are: 1) identify and characterize genes associated with enhanced tolerance to the sugar beet root maggot with focus on the sugar beet taproot; 2) define sugar beet gene promoters for targeting beneficial gene expression to taproot tissues most prone to pest and pathogen attack; and 3) characterize and improve screening of sugar beet for resistance to the sugar beet root maggot by identifying sugar beet root maggot genes that are essential in resistant and susceptible interactions with the host plant.
1b. Approach (from AD-416):
Sugar beet root defense genes incited by the root maggot, a destructive pest of sugar beet, will be characterized using molecular approaches to facilitate screening of sugar beet germplasm for resistance and to devise novel strategies for pest and disease control. Root genes identified as being important in plant defense will be expressed in susceptible plants and screened for enhanced levels of insect pest and phytopathogen resistance. Two resistance genes that preferentially respond to maggot feeding in a moderately resistant germplasm will be studied. One of these genes codes for a serine proteinase inhibitor (PI; BvSTI), and the other for a cell wall polygalacturonase inhibitor (PGIP, BvPGIP). BvSTI is a wound-inducible Kunitz trypsin PI with specificity for the root maggot digestive enzymes that mediate release of essential nutrients from ingested plant tissues. The second gene, BvPGIP, codes for a leucine-rich repeat glycoprotein PGIP associated with cell wall structure and plant defense responses. The role of the sugar beet BvSTI and BvPGIP genes in mediating resistance will be determined by transferring the recombinant genes into sugar beet hairy roots and model whole plants (Nicotiana and Arabidopsis). Genetically modified plants will be bioassayed for resistance to insect pests and phytopathogens. Insect bioassays will include fall armyworm, beet armyworm, or tobacco hornworm larvae and phytopathogens Aphanomyces cochlioides and Fusarium oxysporum that cause root rot in sugar beet. To more precisely target the expression of resistance genes to root cells and tissues most prone to pest and pathogen attack, root tissue-specific and temporal promoters will be identified, cloned, and functionally characterized by differential screening of sugar beet root EST libraries. Cloned promoters will be fused with a reporter gene (GUS) in a plant transformation vector and introduced into sugar beet hairy roots and model whole plants. Various biotic and abiotic stresses such as infestation with insect pests, infection with phytopathogens, mechanical wounding, and treatment with defense response elicitors such as methyl jasmonate and salicylic acid will be used to localize GUS gene expression driven by the selected sugar beet promoters. In complementary studies of insect responses, root maggot genes that are important in resistant and susceptible interactions with the sugar beet root will be identified. Root maggot genes will be profiled, sequenced, and functionally annotated to gain new knowledge of how insects adapt to host plants and surmount host resistance. Expression patterns of identified root maggot genes will be examined to confirm that the selected insect genes are specificity-expressed as a response to feeding on the sugar beet root.
3. Progress Report:
To deliver a safe and secure supply of food to a rising global population, while minimizing harmful impacts on cropping ecosystems, will require that the world’s major crops have increased capacity to resist diseases and insect pests. Sugar beet is an economically important plant that is attacked by numerous insects and pathogens that damage the plant and reduce sugar yields. To improve resistance to insect pests and microbial pathogens, novel resistance traits need to be introduced to sugar beet. We identified a sugar beet gene that is involved in the assembly of the outer protective layer (cell wall) of the root. This outer layer of the plant serves as a barrier and first line of defense against invading insect pests and microbial pathogens. This cell wall gene was cloned, rebuilt for analysis of its function in plants, and reintroduced into sugar beet roots and model plants for further analysis of its role in plant defense capabilities. To date, the cell wall gene has been shown to enhance both pest and fungal resistance in genetically modified plants. The newly gained knowledge will be used to develop plants with stronger outer protective barriers that will improve pest and disease resistance in economically important plants. To effectively steer the production of insecticidal compounds to plant parts most prone to insect attack, characterization of regulatory switches (plant promoters) is needed. We identified several interesting sugar beet switches that have the capability of directing insect and pathogen fighting compounds to the root surfaces, the root itself or to the leaves. These switches were shown to be regulated by insect pest feeding and fungal infections, in addition to mechanical damage of the plant tissues. This information will be used to develop safer approaches of insect control in plants and lead to increased yields and reduced usage of chemical pesticides. Sugar beet root maggot is a destructive pest of sugar beet that is not easily controlled without the use of harmful chemical pesticides. To identify root maggot genes that are important in the interaction of the insect with the resistant and susceptible sugar beet roots, a library of root maggot genes was prepared. These root maggot response genes were sequenced, identified and functionally annotated. Several genes were selected for further analysis in order to gain new knowledge of how insects adapt to host plants and surmount host resistance.
1. Sugar beet resistance genes identified and cloned. Plant diseases and pest problems are responsible for decreases in crop yields. Scientists in Beltsville, Maryland, utilized an enrichment technique to discover and characterize sugar beet root genes involved in pest and disease resistance responses. One of the several genes that were selected for further analysis was cloned and reconstructed for expression in genetically modified plants to determine its role in insect and disease resistance. Several gene switches that exhibit the potential to target the production of insecticidal compounds and/or resistance genes to the root were identified and determined to direct wound, pest and fungal induced expression. Plant biologists will use this information to design new strategies for developing improved plant varieties with enhanced disease and pest resistance that will benefit farmers by increasing yields and the quality and nutritional value of cultivated crops for human consumption.
2. Molecular analysis of sugar beet root maggot genes. A destructive pest of sugar beet, sugar beet root maggot, is found in most areas in the United States where sugar beets are grown and is valued at more than $2 billion. 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 in Beltsville, Maryland, identified sugar beet root maggot genes that are important for disease development in the roots. Plant biologists will use this information for rapid screening of plant varieties for root maggot resistance, functional analysis of resistance genes, and development of improved sugar beet varieties with enhanced root maggot resistance that will benefit farmers by increasing sugar yields for human consumption.
Cingel, A., Savic, J., Vinterhalter, B., Vinterhalter, D., Kostic, M., Smigocki, A.C., Ninkovic, S. 2015. Impaired growth and development of Colorado potato beetle larvae on potato plants overexpressing the oryzacystatin II gene. Transgenic Research. DOI: 10.1007/s11248-015-9873-9.
Vinterhalter, B., Dijana, K.C., Teodora, J., Dejan, P., Ninkovic, S., Smigocki, A.C., Vinterhalter, D. 2015. Gentiana dinarica Beck hairy root cultures and evaluation of factors affecting growth and xanthone production. Plant Cell Tissue And Organ Culture. DOI: 10.1007/s11240-015-0737-z.
Cingel, A., Savic, J., Cosic, T., Raspor, M., Ghalawenji, N., Smigocki, A.C., Ninkovic, S. 2014. Phenotypic performance of transgenic potato (Solanum tuberosum L.) plants with pyramided rice cystatin genes (OCI and OCII). Archives of Biological Sciences, Belgrade. DOI: 10.2298/ABS141201058C.
Li, H., Padmanaban, S., Smigocki, A.C. 2015. Characterization of a Beta Vulgaris polygalacturonase-inhibiting protein: A defense response gene . American Society of Sugar Beet Technologists. http://www.bsdf-assbt.org/assbt/Biennial%20Stuff/assbtbienmtg.htm.
Smigocki, A.C., Li, H., Padmanaban, S., Mongeon, J.E. 2015. Characterization of a Beta vulgaris PGIP defense gene promoter in transgenic plants. Annual Beet Sugar Development Foundation Research Report. http://www.bsdf-assbt.org/assbt/assbt.htm.