INVESTIGATOR: Hibbard, B. E.

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
COLUMBIA, MISSOURI 65211

ESTABLISHMENT AND MOVEMENT OF WESTERN CORN ROOTWORM LARVAE

OBJECTIVES: Specific objectives are to: 1) Determine the effects of competition and resource availability on establishment and subsequent movement of WCR larvae on susceptible corn, 2) Evaluate alternate hosts for their potential importance in the WCR life cycle, 3) Determine establishment success of WCR larvae on host roots of differing maturities, and 4) Determine the behavioral response of WCR larvae to transgenic sources of resistance.

APPROACH: The first field experiment involves infesting individual plants with 100, 200, 400, 800, or 1,600 viable western corn rootworm (WCR) eggs. Sampling for larvae will begin immediately after the first larva is detected by hanging whole root balls in onion bags and allowing them to dry over water pans. In addition, the host status of grasses common to corn fields and adjacent grassy areas will be reexamined in the greenhouse and the field. Giant foxtail, yellow foxtail, green foxtail, baryardgrass, wooly cupgrass, fall panicum, crabgrass, and Johnson grass are among the more common grassy weed species. Each will be evaluated for their ability to support WCR growth and development along with those which were among the better alternate hosts for WCR larvae in previous studies and others common in or near corn. Host establishment will be evaluated by infesting individual plants every 7 days for 10 weeks starting at planting and recovering larvae or adults from plants infested at different growth stages of corn. Lastly, a variety of field and laboratory techniques will be used to evaluate the behavioral response of WCR larvae to transgenic sources of resistance.

NON-TECHNICAL SUMMARY: The introduction of transgenic crops for managing insect pests is one of the most important events in the last 50 years of agriculture. Since more crop land is treated with insecticide for corn rootworms than any other U.S. insect pest, the recent development of this technology for corn rootworm control by several seed companies may offer an environmentally friendly alternative to one of largest markets for insecticides. Maintaining susceptibility to transgenic crops (resistance management) is in the interest of growers, the EPA, and industry. Efforts to develop resistance management plans for prevention of corn rootworm adaptation to transgenic corn cultivars requires knowledge of rootworm larval movement and alternate host utilization that are not currently available. We propose to develop a thorough understanding of corn rootworm larval movement patterns in susceptible corn, transgenic corn, and alternative larval host micro-habitats, which are crucial in deploying effective resistance management strategies. Specifically we propose to: 1) Determine the effects of competition and resource availability on establishment and subsequent movement of WCR larvae on susceptible corn, 2) Evaluate alternate hosts for their potential importance in the WCR life cycle, 3) Determine establishment success of WCR larvae on host roots of differing maturities, and 4) Determine the behavioral response of WCR larvae to transgenic sources of resistance. As a result of this research, timely basic biological information will be generated within a two-year time frame that will assist the EPA in

PROGRESS: 2001/10 TO 2002/09
If registered for use by the Environmental Protection Agency (EPA), the introduction of transgenic corn will offer a viable alternative to insecticides for managing the western corn rootworm, one of the most economically important pests of corn. Maintaining susceptibility to transgenic crops (resistance management) is in the interest of growers, the EPA, and industry, but little is known about many aspects of corn rootworm biology. We have conducted a series of experiments to evaluate larval movement by the western corn rootworm. Previously, two row spacings and two plant spacings were evaluated with a single infestation level to determine if movement occurred. In the summers of 2001 and 2002, infestation levels of 100, 200, 400, 800, 1,600, and 3,200 viable eggs on a central plant were evaluated to determine the effect of egg density and subsequent plant damage on movement after establishment. Movement up to three plants down the row and across a 0.46 m row have been clearly documented after initial establishment. No significant post-establishment larval movement has occurred at lower infestation levels. Larvae apparently move when plants are highly damaged and/or when competition for food exists, but the extent that this movement occurs under normal field infestations (without a single point source) remains unclear. Another aspect of rootworm biology related to resistance management for which little information is available is the level of importance that alternate hosts play in the life cycle of the western corn rootworm. Previously, we evaluated 28 species of major grassy weeds for their host status with western corn rootworm larvae in the greenhouse. Twenty-one of the species evaluated supported western corn rootworm larval growth at least to the third instar and most of these grasses can be found in or near corn fields. In field experiments in 2001 and 2002, we evaluated grassy weed species by themselves, with isoline corn and with transgenic corn. We demonstrated that adults were produced from five of nine grassy weed species evaluated, and more adults were produced from a combination of grass and transgenic corn than either the transgenic corn or the grass alone.

IMPACT: 2001/10 TO 2002/09
Grasses in, near, and perhaps even distant from corn may play more of a role in the rootworm life cycle than most rootworm entomologists would have guessed, implying that resistance management models for corn rootworm adaptation to transgenic corn (computer simulations at this point) may need to have some of their assumptions adjusted.

PUBLICATIONS: 2001/10 TO 2002/09
Hibbard, B.E., D.P. Duran, M.R. Ellersieck, and M.M. Ellsbury. 2003. Post-establishment movement of western corn rootworm larvae (Coleoptera: Chrysomelidae) in Central Missouri corn. J. Econ. Entomol. (Accepted, In Press).

PROJECT CONTACT:

Name: HIBBARD, B. E.
Phone: 573-882-6281
Fax: 573-882-7850
Email: hibbard@missouri.edu

Item No. 10 of 13

INVESTIGATOR: Tipton, P. A.

PERFORMING INSTITUTION:
BIOCHEMISTRY
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

SOYBEAN URATE OXIDASE AND THE UREIDE PATHWAY

OBJECTIVES: To trap and identify intermediates that form during the catalytic cycle of the enzyme urate oxidase, and to clone the gene encoding hydroxyisourate hydrolase from soybean root nodules.

APPROACH: To identify intermediates in the urate oxidase reaction, rapid-mixing chemical quench techniques are being applied. Components in the quenched reaction mixtures are purified by HPLC and identified using mass spectrometry. Cloning of the gene encoding hydroxyisourate hydrolase is being accomplished with PCR-based techniques using primers that have been designed based on the amino acid sequence of tryptic fragments of purified hydroxyisourate hydrolase.

NON-TECHNICAL SUMMARY: Nitrogen is a limiting nutrient for most crops; legumes such as soybeans occupy a special place in agriculture because they possess the ability to convert atmospheric nitrogen into ammonia. The metabolic pathway by which the ammonia is converted into organic compounds that can be used by the plant to support its growth is called the ureide pathway. Nitrogen fixation is an energetically costly process for the plant, and a worthwhile goal for agricultural research is to enhance the efficiency of the process by which nitrogen becomes available to the plant in usable form. A prerequisite for doing so, however, is gaining a detailed understanding of the ureide pathway and its component enzymes. One of the enzymes in the ureide pathway is urate oxidase, which catalyzes the conversion of urate to 5-hydroxyisourate. It is now known how 5-hydroxyisourate is converted to allantoin, which is the metabolite that the plants transport from the roots, where nitrogen fixation and the urate oxidase reaction occur, to the stem and leaves, where the nitrogen is used in amino acid biosynthesis. One current goal is to characterize 5-hydroxyisourate hydrolase, which may be a hitherto unrecognized constituent of the ureide pathway. A second goal is to continue mechanistic studies of urate oxidase by isolating and identifying intermediates formed during the catalytic cycle. These studies are leading to an understanding of how urate oxidase can operate in the absence of any of the cofactors that typically mediate dioxygen-dependent enzymatic reactions.

PROGRESS: 2001/10 TO 2002/09
Experiments conducted in the past year demonstrated that allantoin produced by the action of the ureide pathway in soybean root nodule slices is optically active, which indicates that every step in the pathway must be enzyme-catalyzed. Therefore, there remains at least one more enzyme in the pathway to be discovered and characterized. We determined the organellar and subcellular localization of hydroxyisourate hydrolase, and defined the temporal expression of its gene in relation to onset and cessation of nitrogen fixation. All of the data are consistent with hydroxyisourate hydrolase participating in the ureide pathway. Experiments were conducted to characterize the chemical mechanism of hydroxyisourate hydrolase. Sequence alignments suggested that it shares considerable homology with well-characterized retaining glycosidases, which utilize glutamate residues at the active site to mediate substrate hydrolysis. We identified analogous glutamate residues in hydroxyisourate hydrolase and showed by site-directed mutagenesis that they play critical roles in the catalytic reaction. The addition of anionic nucleophiles to the reaction medium accelerated the rate of the enzymatic reaction, which suggests that it proceeds via formation of an enzyme-substrate covalent intermediate. We conducted mechanistic studies of urate oxidase; specifically, rapid-mixing chemical quench experiments were performed to characterize the timecourse for urate dianion formation during the catalytic cycle. Site-directed mutagenesis was used to identify the amino acid residues required for substrate deprotonation.

IMPACT: 2001/10 TO 2002/09
Our recent data strongly suggest that hydroxyisourate hydrolase is a legitimate constituent of the ureide pathway, and clearly demonstrate that another, as yet unidentified enzyme, participates in the pathway as well. Thus, it is clear that this important metabolic pathway remains poorly characterized. It is anticipted that a better understanding of the ureide pathway is required for efforts to improve the efficiency of nitrogen utilization by soybeans. The mechanistic studies of urate oxidase continue to define the parameters and characteristics of cofactor independent oxygen-mediated enzymatic reactions.

PUBLICATIONS: 2001/10 TO 2002/09
No publications reported this period

PROJECT CONTACT:

Name: TIPTON, P. A.
Phone: 573-882-7968
Fax: 573-884-4812
Email: tiptonp@missouri.edu

Item No. 11 of 13

INVESTIGATOR: Mcmullen, M. D.; Lee, E. A.; Byrne, P. F.; Snook, M. E.; Wiseman, B. R.; Widstrom, N. N.

PERFORMING INSTITUTION:
PLANT GENETICS
AGRICULTURAL RESEARCH SERVICE
COLUMBIA, MISSOURI 65211

GENETIC CONTROL OF CORN EARWORM RESISTANCE FACTORS IN MAIZE

OBJECTIVES: 9701357. Determine the role of known genes in the flavonoid pathway in controlling maysin synthesis. Determine the relationship of expression of flavonoid genes with QTL controlling maysin synthesis and corn earworm resistance. Relate information gained to enhance corn earworm resistance in maize lines.

APPROACH: Use QTL analysis to determine the role of known genes in the flavonoid pathway in controlling maysin synthesis. Use insect bioassays to correlate maysin levels with antibiosis. Use gene expression studies to determine the role of transcription activation in determining QTLs for maysin synthesis.

PROGRESS: 1997/10 TO 1998/09
With this project we will continue studies to determine the relationship of specific genes in the flavonoid pathway with QTLs for flavone synthesis and corn earworm (CEW) resistance. This summer we initiated experiments with five new populations with the following specific objectives. The population (T218 x GT119)F2 to determine the genetic basis of the salmon silk phenotype and to study epistatic interaction of the p1 and sm1 loci. The population (W23al x GT119)F2 to determine the effect of a block in the 3-deoxyanthocyanin pathway on flavone synthesis and CEW resistance. The population (Mo1W & Mo20W x A619)F2s to study flavone synthesis and CEW resistance directed by pl-wwb alleles. Two populations are being studied to determine the relationship of QTLs for flavone synthesis and levels of gene expression of genes of the flavonoid pathway, the population (GT114 x GT119)F2 to study activation of the flavone pathway by variation at p1, and the population (GE37 x Mp464)F2 to study the relative expression of the two chalcone synthase genes, c2 and whp1. During the summer of 1997 silks were collected from all these populations and genetic and chemical analyses are in progress.

PUBLICATIONS: 1997/10 TO 1998/09
1. MCMULLEN, M.D., BYRNE, P.F., SNOOK, M.E., WISEMAN, B.R, LEE, E.A., WIDSTROM, N.W., and COE, E.H. (1998) Quantitative trait loci and metabolic pathways. Proc. Natl. Acad. Sci. USA 95:1996-2000.
2. BYRNE, P. F., MCMULLEN, M. D., WISEMAN, B. R., SNOOK, M. E., MUSKET, T. A., THEURI, J, M., WIDSTROM, N. W., and COE, E. H. (1998) Maize silk maysin concentration and corn earworm antibiosis: QTLs and genetic mechanisms. Crop Sci. 38:461-.
3. LEE, E.A., BYRNE, P.F., MCMULLEN, M.D., SNOOK, M.E., WISEMAN, B.R., WIDSTROM, N.W., and COE, E.H. (1998) Genetic mechanism underlying apimaysin and maysin synthesis and corn earworm antibiosis in maize (Zea mays L.). Genetics 149:1997-2006.

Item No. 12 of 13

INVESTIGATOR: Stuart, M. K.

PERFORMING INSTITUTION:
MICROBIOLOGY & IMMUNOLOGY
KIRKSVILLE COLL OF OSTEO MED
KIRKSVILLE, MISSOURI 63501

ELONGATION FACTOR 1-ALPHA AS A POTENTIAL TARGET FOR DISRUPTION OF INSECT METABOLISM

OBJECTIVES: 9803871. The primary goal of this project is the production of monoclonal antibodies that can inactivate eucaryotic translation elongation factor-1a(EF-1a) from the fall armyworm, Spodoptera frugiperda. This is the first step toward the long-range goal of generating biopesticides capable of inhibiting protein synthesis in target pests, a goal that will be achieved by engineering baculoviruses to express EF-1a-specific single-chain antibodies within the cytoplasm of host cells.

APPROACH: Monoclonal antibodies specific for EF-1a will be generated from mice immunized with 1) synthetic peptides that are based on amino acid sequences unique to S. frugiperda EF-1a, and 2) whole-molecule EF-1a isolated from the S. frugiperda cell line, Sf21. An antibody shown to recognize native EF-1a by its ability to immunoprecipitate the nondenatured enzyme from cellular homogenates will be further examined for its effects on protein synthesis in cell-free in vitro translation systems established from Sf21 and BTI-TN-5BI-4 cells, the latter derived from the cabbage looper, Trichoplusia ni. The ability of antibodies to inhibit protein synthesis will be determined by adding luciferase mRNA and purified antibody to translation-competent insect cell lysates pre-treated with micrococcal nuclease to remove endogenous mRNA, and then examining new translation products for luciferase protein. Incorporation of biotinylated lysines into newly synthesized luciferase will be detected by a non-radioactive colorimetric method after the translation products have been subjected to SDS-PAGE and transferred to nitrocellulose.

PROGRESS: 1998/09 TO 2001/09
Our LONG-RANGE GOAL is to enhance the baculovirus as a microbial insecticide by engineering it to express single-chain antibodies capable of inactivating essential enzymes in insect cells. Our first target for this technology is elongation factor-1 alpha (EF-1a), an enzyme necessary for protein synthesis. The SPECIFIC AIMS of the current project were to: 1) PRODUCE MURINE MONOCLONAL ANTIBODIES (MABS) TO EF-1a FROM THE FALL ARMYWORM, SPODOPTERA FRUGIPERDA. This goal was met by immunizing mice with EF-1a passively eluted from SDS-PAGE gels loaded with homogenates of Sf21 cells, and fusing the spleen cells to myelomas. Seventy-five hybridoma lines secreting MABs specific for EF-1a were obtained. The identity of the antigen recognized by the MABs was confirmed by internal amino acid sequence analysis, and by the antigen's cross-reactivity with a monospecific rabbit antiserum generated to EF-1a from Dictyostelium discoideum. Four MABs were chosen for further analysis. All four antibodies reacted with EF-1a in eggs and first through fifth instars of the fall armyworm, but did not recognize EF-1a from a human cell line. 2) IDENTIFY THOSE MABS THAT RECOGNIZE EF-1a IN ITS NATIVE CONFORMATION. This goal was met, as evidenced by the ability of the MABs to immunoprecipitate EF-1a from homogenates of Sf21 cells prepared in a non-denaturing buffer. 3) ESTABLISH A CELL-FREE IN VITRO TRANSLATION SYSTEM DERIVED FROM SF21 CELLS. This goal was met, as evidenced by autoradiographic analysis of acid-precipitable proteins that incorporated [35S]-methionine during synthesis by Sf21 cell lysates. 4) IDENTIFY THOSE MABS WITH THE ABILITY TO INHIBIT PROTEIN SYNTHESIS IN AN INSECT-DERIVED CELL-FREE TRANSLATION SYSTEM. This goal was met by adding purified MABS to the Sf21-derived in vitro translation system and measuring changes in protein synthesis by scintillation. Of the four EF-1a-specific MABs tested, three significantly inhibited incorporation of [35S]-methionine into acid-precipitable proteins (and thus protein synthesis) compared to a negative control antibody matched for isotype (P<0.001, one-way ANOVA; followed by Dunnett's test, P<0.05). In future studies, hybridomas that secrete inhibitory MABs will be used as the source of DNA used to create recombinant baculoviruses secreting single-chain antibodies specific for EF-1a. Of the three hybridoma lines secreting inhibitory MABs identified during the current study, two are particularly well suited for the next phase of the project involving production of recombinant viruses. MABs 7D6 and 7G3 demonstrated no cross-reactivity for High Five (BTI-TN-5B1-4) cells derived from the cabbage looper, Trichoplusia ni. Such species specificity is fortuitous because it will allow the High Five cell line to be used to propagate recombinant baculoviruses to control fall armyworm infestations. Since the antibodies do not bind to EF-1a from High Five cells, there should be no toxic effects to the culture system that would prevent the maturation of infectious virions containing the antibody genes.

IMPACT: 1998/09 TO 2001/09
The baculovirus is a safe alternative to chemical insecticides for controlling pest insects because it does not infect fish, birds, or mammals. However, the baculovirus often requires a week or more to kill insects under field conditions. Through recombinant DNA technology, we intend to equip the baculovirus with genes encoding antibodies that can rapidly shut down insect cell metabolism, leading to more rapid insect death or cessation of feeding, thus enhancing the value of the baculovirus as a microbial pesticide. In the current study, we have produced antibodies that are capable of inhibiting protein synthesis in insect cells. The genes encoding these antibodies are good candidates for use in producing the enhanced baculoviruses.

PUBLICATIONS: 1998/09 TO 2001/09
Stuart MK and Chamberlain NR (2001): Monoclonal antibodies to elongation factor-1a inhibit in vitro translation in lysates of Sf21 cells. Submitted to Archives of Insect Biochemistry and Physiology (MS #01-061).

Item No. 13 of 13