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Research Project: Molecular Technology for Developing Durable Pest and Pathogen Resistance in Sugar Beet

Location: Molecular Plant Pathology Laboratory

2018 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:
This is the final report to 8042-21220-231-00D entitled Molecular Technology for Developing Durable Pest and Pathogen Resistance in Sugar Beet. The new project 8042-21220-233-00D was recently approved and is entitled "Emerging Biotechnologies for Developing Improved Pest and Pathogen Resistant Sugar Beet." 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 food crop, being one of only two plant sources from which more than one third of global raw sugar is economically produced. Its annual value is triple that of sugarcane in the U.S. alone. Sugar beet 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 sugar beet genes with a role in resistance, among them a gene that is involved in the assembly of the root cell wall. Cell walls serve as a barrier and first line of defense against invading insect pests and microbial pathogens. We demonstrated that overproduction of this gene’s protein product in genetically modified sugar beet roots and model plants increased their tolerance to several fungal pathogens that cause yellows diseases and root and stem rot in sugar beet and hundreds of other vegetable crops. Enhanced fungal resistance to Rhizoctonia solani and several Fusarium species was demonstrated. We also demonstrated for the first time that this gene concomitantly enhanced resistance to insect pests, including beet and fall armyworm, pests that attack sugar beet and a multitude of other commercially important crops. This newly discovered sugar beet gene with demonstrated broad dual resistance to both fungal pathogens and insect pests will be used to develop plants with enhanced natural resistance to pests and microbial diseases. Limited information is available on the molecular level on how insects evolve adaptive mechanisms to overcome host resistance and develop tolerance to many insecticides. Sugar beet root maggot is a destructive pest of sugar beet that is not easily controlled without the use of harmful chemical pesticides. Many of the pesticides are being taken off the market which may lead to devastating yield losses for the sugar beet growers. We are addressing the root maggot problem using biotechnology. We identified over 300 root maggot genes that are important in the interaction of this insect with the resistant or susceptible sugar beet host. These root maggot response genes were sequenced and functionally annotated. Among the profiled genes, we identified insect genes that are critical in physiology, development, regulation and cellular processes, as well as genes responsive to environmental conditions. Several genes were selected as possible targets involved in insect-host interactions. Functional analysis of the root maggot genes by cutting edge genome editing technology is being pursued to gain new knowledge of how insects adapt to host plants and surmount host resistance.

4. Accomplishments
1. Sugar beet resistance genes identified, cloned and functionally defined. Plant diseases and pest problems are responsible for decreases in crop yields; therefore, ARS 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 introduced into genetically modified plants to determine its role in insect and disease resistance and the findings revealed a new gene structure that is uniquely associated with sugar beet. ARS scientists in Beltsville, Maryland, demonstrated that this gene provides broad resistance to fungal pathogens and insects that attack sugar beet and other commercially important crops. Discovery of these genes by the Beltsville scientists will be used by plant biologists for designing new theories and hypotheses for improving and incorporating broader disease and pest resistance. Plant biologists will develop improved plant varieties with enhanced disease and pest resistance that 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. ARS scientists in Beltsville, Maryland adapted an enrichment technique for discovering root maggot genes. ARS scientists first identified more than 300 root maggot genes and demonstrated that they are important for immunity, insecticide resistance, and for interaction with a resistant sugar beet plant. This new knowledge of root maggot genes can help plant biologists improve the plants’ pest and disease resistance. Plant biologists will use this information for rapid screening of plant varieties for root maggot resistance, for functional analysis of resistance genes, and to develop improved sugar beet varieties.

Review Publications
Smigocki, A.C., Mongeon, J., Savic, J. 2017. Sugar beet proteinase inhibitor (BvSTI) gene promoter is regulated by insects and wounding in transgenic Nicotiana. Journal of Sugar Beet Research. 55:1-28.

Smigocki, A.C., Li, H., Mongeon, J.E., Braza, R., Bruccoleri, R. 2018. Novel use of Beta vulgaris lipid biosynthesis genes for disease resistance and production of biofuels. Annual Beet Sugar Development Foundation Research Report.

Li, H., Smigocki, A.C. 2018. Sugar beet polygalacturonase-inhibiting proteins with 11 LRRs confer Rhizoctonia, Fusarium and Botrytis resistance in Nicotiana plants. Physiological and Molecular Plant Pathology. 102:200-208.