Search USDA's Current Research Information System (CRIS) Database


Item No. 1 of 12

ACCESSION NO: 0186038 SUBFILE: CRIS
PROJ NO: KS9184 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35302-09983 PROPOSAL NO: 2000-02970
START: 01 DEC 2000 TERM: 30 NOV 2003 FY: 2001 GRANT YR: 2001
GRANT AMT: $290,000

INVESTIGATOR: Passarelli, A. L.

PERFORMING INSTITUTION:
BIOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

ROLE OF BACULOVIRUS EXPRESSION FACTORS IN HOST RANGE

OBJECTIVES: Study structure-function relationships of late expression factor-8, an Autographa californica multinucleocapsid nucleopolydrovirus (AcMNPV) gene encoding a DNA-directed RNA polymerase subunit. Identify late expression factors (lefs) involved in host range and determine the protein regions required for late gene expression and host range determination.

APPROACH: Plasmids with mutations that alter a sequence motif within lef-8 that is also found in the b or b' subunit of DNA-directed RNA polymerases in animals, plants, eubacteria and archaebacteria will be tested for their functionality in previously developed transient gene expression assays. Deletion analysis will delineate the other region(s) necessary for function. lefs of closely related and more distant baculoviruses will be substituted for the AcMNPV counterparts and tested in well-characterized activity assays. Chimeras between homologous genes will be constructed to functionally define critical regions.

NON-TECHNICAL SUMMARY: The long-term goal of this research is to identify and characterize genetic host range determinants in order to successfully manipulate baculovirus host restrictions and improve their pesticide efficacy. A number of genes (late expression factors or lefs) of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) are necessary for late gene transcription and/or DNA replication.

PROGRESS: 2002/01 TO 2002/12
We have completed the mutagenesis of the baculovirus AcMNPV late expression factor-8 gene, the largest subunit of the virus-encoded RNA polymerase required for the specific transcription of late-stage genes. We found that regions throughout this subunit were important for function, highlighting the complex and numerous interactions that occur during transcription of viral genes. We are currently defining these interactions in vivo and in vitro. We are in the process of determining the factors necessary for productive baculovirus infections in permissive and non-permissive hosts by comparing gene activities of potential host range genes from baculoviruses with different host ranges. The role of these factors in genetic recombination events is also being evaluated. To this end, we developed an assay that allowed the identification of viral genes necessary for homologous recombination. The ability of these viruses to infect insect hosts may be closely linked to their ability to replicate and repair their genomes using both viral and host enzymes.

IMPACT: 2002/01 TO 2002/12
Industry and agriculture would benefit from baculoviruses with a wider host range to effectively control pests. In order to accomplish this successfully, we are identifying and characterizing baculovirus host range genes. Recombination in baculoviruses will help understand genetic heterogeneity and speciation that are important in the assessment of bioinsecticides. Overall, this study will aid in developing a safe food supply and improve human health.

PUBLICATIONS: 2002/01 TO 2002/12
Crouch, E. A. and A. L. Passarelli. 2002. Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus. J. Virol. 76:9323-9334.

PROJECT CONTACT:

Name: Passarelli, A. L.
Phone: 785-532-3195
Fax: 785-532-6653
Email: lpassaar@ksu.edu

Item No. 2 of 12

ACCESSION NO: 0170306 SUBFILE: CRIS
PROJ NO: KS9223 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 95-37302-1837
START: 01 JUL 1995 TERM: 30 JUN 1999 FY: 1999 GRANT YR: 1995
GRANT AMT: $120,000

INVESTIGATOR: Reeck, G. R.; Reese, J.

PERFORMING INSTITUTION:
BIOCHEMISTRY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

APHID PECTINASES IN INSECT-PLANT INTERACTIONS AND BIOTYPE EVOLUTION

OBJECTIVES: 9502102. Our objectives are: to isolate and determine the nucleotide sequencesof cDNA genomic clones that encode greenbug polygalacturonase and pectinmethylesterase; and to examine variation in pectinase gene expression in aphids that differ in phytotoxicity.

APPROACH: We will screen an aphid cDNA library with cDNA clones that encode rice weevil (Sitophilus oryzae) polygalacturonase and pectinmethylesterase or use PCR-based methods to obtain greenbug cDNA clones of interest. These will be used in turn to screen a greenbug genomic library. Northern analysis and, potentially, sequencing of cDNA clones will be used to assess quantitative and qualitative differences in pectinase genes in other aphid species.

PROGRESS: 1995/07 TO 1999/06
We continued our investigations of the interpretation of plate assays of mixtures of pectinases (polygacturonase and pectinmethylesterase). To re-set the scene, when greenbugs inject saliva into pectin/agar plates, one sees zones of clearing with intense red staining (halos) around the outside, after the plates are stained with ruthenium red. Using extracts from other insects that produce polygalacturonase and pectinmethylesterase and mixtures of purified rice weevil polygalacturonase and pectinmethylesterase, and by studying the staining patterns as a function of pH, we can both explain the peculiar staining patterns and estimate the pH optima of the two enzymes. The characteristic set of patterns (red circles only at relatively high pH; zones of clearing with red halos at intermediate pH; and simple zones of clearing at lower pH) are due to the differences in pH optima of the pectinmethylesterase (relatively high) and the polygalacturonase (relatively low). This relationship holds in all insects studied. The difference in the pH optima varies, however. For instance, the optima of the two enzymes appear to be farther apart in the greenbug than they are in the rice weevil. The physiological significance of the difference in pH optima or the variation in that difference from species to species is not yet clear.

IMPACT: 1995/07 TO 1999/06
In the long term, studies of enzymes, including pectinases, in salivary secretions of the greenbug could identify targets for genetically engineered increases in plant resistance to this insect.

PUBLICATIONS: 1995/07 TO 1999/06
Shen, Z., Manning, G., Reese, J.C., and Reeck, G.R. 1999. Pectin Methylesterase from the Rice Weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae): Purification and Characterization, Insect Biochem. Mol. Biol. 29: 209-214.


Item No. 3 of 12

ACCESSION NO: 0184849 SUBFILE: CRIS
PROJ NO: KS9428 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 96-35312-5314 PROPOSAL NO: 2000-04000
START: 01 JUL 2000 TERM: 30 APR 2001 FY: 2001 GRANT YR: 1996
GRANT AMT: $18,443

INVESTIGATOR: Zolnerowich, G.

PERFORMING INSTITUTION:
ENTOMOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

REVISION OF NEARCTIC METAPHYCUS, WITH A PHYLOGENETIC ANALYSIS OF APHYCINI

OBJECTIVES: The first objective is a revision of Nearctic species of Metaphycus. The second objective is a prelininary phylogenetic analysis of Aphycini, which includes Metaphycus and additional genera.

APPROACH: Species of Metaphycus are important in biological I control because they are solitary or gregarious parasites of the superfamily Coccoidea. Species attack economically important genera such as Coccus,Philephedra, and Saissetia. Nearctic Metaphycus are difficult to identify due to inadequate descriptions and lack of keys. The revision treats at least 35 described species, with new species to be described. A comparative methodology utilizing morphological characters as well as previously unexplored characters will be utilized. Descriptions, illustrated keys to males and females, and distribution and host information will be prepared. The revision and keys will include exotic species of Metaphycus established as part of biological control programs. Evidence suggests the tribe is not a single lineage. Monophyly of Aphycini will be examined and morphological data will be analyzed with computer programs that employ parsimony analysis. Relationships will be inferred by possession of synapomorphies.

NON-TECHNICAL SUMMARY: Encyrtidae represent one of the two families with the highest number of introductions and establishments in attempts to control Homoptera or other pests. Understanding of the systematics of encyrtid genera such as Metapycus important to bio- logical control has increased tremendously, but without the revisionary work that results in keys to identification, an understanding of species limits, and insights into the phylogeny of a group. Lack of identification tools and an understanding of such groups hinders their use in biological control.

PROGRESS: 2000/07 TO 2001/04
Specimens continued to be sorted to various species. A number of type specimens were sent to the USDA Systematic Entomology Laboratory (Washington, DC) for examination by J. S. Noyes, another systematist working on Metaphycus from other parts of the world. Funding was redirected to the purchase of equipment that would facilitate the work. A Nikon DXM1200 digital camera, a Dell computer, and Syncroscopy Auto-Montage software were purchased. The digital camera is used to produce high quality digital images of insect specimens or parts of specimens for research, and illustrations for publications. The software is a specialized program expressly designed to be used in conjuction with digital photography through microscopes. Because depth-of-field is a problem inherent in micro-and macro-photography, this software allows multiple photographs to be taken at different focal points on the speciment, and then digitally combined into a single, in-focus image. Measurements important to research, such as length, width, area, etc., can be done with the software. The camera and software are controlled through the computer. Other researchers have made use of this new optical system, including photographing images of ovariole and oviduct development in pest species of Noctuidae, studying the mating behavior of long-horned beetles, and capturing images as part of a monograph on Costa Rican braconid wasps. Research in progress on Metaphycus continues.

IMPACT: 2000/07 TO 2001/04
Improved understanding of the systematics of natural enemies allows them to be used more effectively in biocontrol programs, and to provides a better understanding of their role in natural and agricultural systems. Given the advantages of biological control, systematics research in this area is an important component of Integrated Pest Management.

PUBLICATIONS: 2000/07 TO 2001/04
No publications reported this period

PROJECT CONTACT:

Name: Zolnerowich, G.
Phone: 785-532-3799
Fax: 785-532-6258
Email: gzolnero@ksre.ksu.edu

Item No. 4 of 12

ACCESSION NO: 0180450 SUBFILE: CRIS
PROJ NO: KS9621 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 98-35303-6446
START: 15 SEP 1998 TERM: 31 MAR 2001 FY: 2001 GRANT YR: 1998
GRANT AMT: $145,000

INVESTIGATOR: White, F. F.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

TRANSCRIPTION ACTIVATION BY AVRXA10 AND AVRXA7 IN RESISTANCEAND PATHOGENICITY

OBJECTIVES: 9802109. The objectives of the research are to determine the effects of substitutions in the amino acid residues of the acidic transcriptional activation domain of AvrXa10 on activation and avirulence on rice plants. A second objective is to determine the effects of the above substitutions in a closely related protein, AvrXa7, on the virulence properties of the protein.

APPROACH: Standard recombinant DNA techniques will be used to alter the DNA coding sequence of the genes for the proteins. The two principal techniques will be oligonucleotide-directed mutagenesis and polymerase chain reaction (PCR). Once constructed, the genes will be introduced into Xanthomonas oryzae pv. oryzae, and the strains will be tested for their ability to elicity resistance or disease on specific rice cultivars.

PROGRESS: 1998/09 TO 2001/03
Bacterial avirulence genes control the elicitation of resistance in plants if the plant contains the corresponding gene for resistance. Avirulence genes also, in some cases, control the degree of virulence or pathogenicity of the bacterial strain. The genes avrXa7 and avrXa10 are members of the avrBs3 avirulence gene family. These two genes contain features in the encoded protein that have the characteristics of plant transcription factors including an acidic transcriptional activation domain in the carboxyl terminal end of the protein product. We characterized the activation domain of AvrXa10 by mutagenesis of the avrXa10 gene and determined the requirement of the domain for avirulence activity and determined the requirement of the domain for the avirulence and pathogenicity effects of AvrXa7. Corroborating evidence for the transcriptional activator role of the proteins was obtained from domain swapping experiments using heterologous activation domains and protein localization experiments.

IMPACT: 1998/09 TO 2001/03
The studies afforded a better understanding of the mechanisms of plant resistance to disease and provided insight into new strategies for control of diseases.

PUBLICATIONS: 1998/09 TO 2001/03
No publications reported this period


Item No. 5 of 12

ACCESSION NO: 0182237 SUBFILE: CRIS
PROJ NO: KS9638 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 99-35300-7702 PROPOSAL NO: 1999-01395
START: 01 SEP 1999 TERM: 31 AUG 2002 FY: 2001 GRANT YR: 1999
GRANT AMT: $120,000

INVESTIGATOR: Trick, H. N.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

MAIZE RESISTANCE GENES EXPRESSED IN TRANSGENIC CEREALS

OBJECTIVES: Demonstrate that over-expressed Rp1-D gene in a susceptible maize background will cause an increased level of resistance against maize leaf rust. Produce transgenic wheat expressing the maize Rp1-D gene. Determine the functionality of the maize Rp1-D gene in transgenic wheat.

APPROACH: Transgenic maize and wheat plants will be produced over-expressing the maize leaf rust resistance gene (Rp1-D). A full-length genomic Rp1-D gene as well as an over-expressed, ubiquitin driven coding sequence of Rp1-D will be introduced using the particle gun. Putative transgenic plants recovered will be confirmed using standard molecular approaches including PCR, Southern-blot, Northern-blot, Western-blot analyses. For western-blot analysis, synthetic peptides are being used to construct Rp1-D specific antibodies. Five to fifteen independently transformed T0 maize and wheat from each construct will be selected for further analyses. T1 and T2 progeny will be grown and gene expression and stability will be assessed. Transgenic maize plants will be challenged with common and southern leaf rust to test for disease resistance. Transgenic wheat plants will be challenged with at least four pathotypes of Puccinia recondita (leaf rust) at the four leaf and adult stage. Resistance may be expressed as an obvious difference in reaction type, as the race specific effects often are. If not, quantitative levels of resistance will be reported, especially at the adult-plant stage. This may be quantified either as a delay in pustule development or a reduced number of pustules.

NON-TECHNICAL SUMMARY: The goal of the proposed research is to understand the mechanisms involved in the broad-spectrum resistance of the genes in the maize rp1 locus toward maize leaf rust and to determine whether Rp1 genes can function in other cereals such as wheat. This proposed research is expected to demonstrate that over-expressed Rp1-D gene in maize is responsible for resistance against maize leaf rust and Rp1-D has a similar result against wheat leaf rust when the gene is introduced into wheat. A full-length genomic sequence of the recently cloned Rp1-D gene of maize, as well as the over-expressed coding region, will be introduced into maize and wheat via particle bombardment. Based on the molecular analyses and Rp1-D expression levels, 5-10 transgenic maize and wheat lines will be selected for pathogen inoculations. Transgenic maize plants will be challenged with maize leaf rust and southern leaf rust. The transgenic wheat will be challenged with wheat leaf rust. Plants will be scored based on the number and the size of rust pustules as well as a delay in pustule formation. This proposed research should answer fundamental questions pertaining to the scope of the RP1 resistance genes. Understanding the mechanisms involved in the broad-spectrum resistance of the genes in the maize rp1 locus toward maize leaf rust and to determining whether Rp1 genes can function in other cereals such as wheat is a priority of this research. If successful this research will provide a new source of resistance in wheat against rust not obtainable by conventional breeding techniques.

PROGRESS: 1999/09 TO 2002/08
The Rp1 complex of maize carries a family of nucleotide-binding site, leucine-rich repeat genes (NBS-LRR) which control resistance to common leaf rust (Puccinia sorghi). Two of these genes were analyzed for their resistance efficiency in transgenic cereals. The Rp1-D gene promotes race-specific rust resistance while the recombinant Rp1-D*21 gene was thought to confer a non-specific reaction to Puccinia isolates resulting in a necrotic spotting lesion mimic phenotype. A genomic clone of the Rp1-D gene with its native regulatory elements and a construct with the maize ubiquitin promoter were independently transformed into susceptible maize lines via biolistics. The resulting T0 plants were crossed one or more times to the maize lines HII and H95 to identify stably expressing rust resistance. Different intensities of race-specific rust resistance were observed in several independent transgenic genotype families. It also seems likely that different phenotypes can be associated with expression levels. Two segregating maize lines expressing a copy of the Rp1-D*21 gene were constructed and both exhibited the non-specific necrotic spotting phenotype. Transgenic corn plants challenged with the wheat leaf rust (Puccinia triticina) did elicit a necrotic phenotype suggesting that the gene does recognize the wheat pathogen. Both the Rp1-D and the Rp1-D*21 gene were also transformed into wheat to analyze their utility and functionality in evolutionary divergent cereals. Sixteen independent Rp1-D transgenic lines and 24 of Rp1D-21 were analyzed up to the T4 generation and approximately 30% of them showed stable transcription of the transgenes. None of these lines, however, exhibited any noticeable increase in resistance to wheat leaf rust (P. triticina) or any chlorotic or necrotic spotting. Although RT-PCR results confirmed expression in transgenic wheat, detection of the transgene by northern blots were unsuccessful. Conclusions from these experiments suggest that the downstream pathways involved in triggering a resistant response were not activated by either low protein levels or that the RP1 proteins are unable to interact with the wheat signaling pathway.

IMPACT: 1999/09 TO 2002/08
This project may result in increased wheat leaf rust resistance, result in yield stabilization in pathogen infected areas, and facilitate breeding programs by introducing new traits to elite cultivar lines. Although attempts from these experiments were unsuccessful, it may be possible to perform domain swapping experiments with wheat resistance genes with the RP-1 family members to achieve this goal.

PUBLICATIONS: 1999/09 TO 2002/08
No publications reported this period

PROJECT CONTACT:

Name: Trick, H. N.
Phone: 785-532-6176
Fax: 785-532-5692
Email: trick@plantpath.ksu.edu

Item No. 6 of 12

ACCESSION NO: 0186415 SUBFILE: CRIS
PROJ NO: KS9662 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35319-09851 PROPOSAL NO: 2000-02698
START: 01 NOV 2000 TERM: 31 OCT 2003 FY: 2001 GRANT YR: 2001
GRANT AMT: $300,000

INVESTIGATOR: White, F. F.; Johnson, L. B.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

CHARACTERIZATION OF THE PATHOGENICITY PROTEIN AVRXA7

OBJECTIVES: 1. Determine if AvrXa7 has dsDNA-binding properties. 2. Identify plant DNA sequences that bind AvrXa7, if evidence for sequence specificity is found. 3. Characterize the effects of AvrXa7 expression in the plant on host gene expression (gene profiling) and physiology.

APPROACH: The ability of Xanthomonas oryzae pv. oryzae to cause the disease bacterial blight of rice depends on a functional type-III secretion system, which in phytopathogenic bacteria is commonly known as the Hrp system. In the absence of Hrp function, X. oryzae pv. oryzae causes no visible symptoms on the host plant. The Hrp secretion pathway is required for pathogenicity in a variety of Gram negative plant pathogenic bacteria and controls the secretion of a set of bacterial virulence and avirulence factors during the growth of the bacterium in the host plant. Some evidence suggests that the factors are involved in overcoming the general resistance mechanisms of the host. Plants, as part of their surveillance and defense system, have evolved mechanisms that, among other compounds, commonly recognize type-III-dependent virulence factors of pathogens and trigger a HR. Avirulence genes encode proteins that have been recognized by the host surveillance and defense systems. The Hrp-dependent gene avrXa7 encodes the protein AvrXa7, which is secreted to plant cells and triggers a HR in rice cultivars containing the resistance gene Xa7. AvrXa7 is also required for full virulence in strain PXO86 of X. oryzae pv. oryzae. We are trying to determine the mechanism of AvrXa7 function in virulence. The avrXa7 gene is a member of the avrBs3 gene family and, as such, has interesting features including a repetitive structural domain for R-gene specificity, nuclear localization motifs, and a transcriptional activation domain. The features provide evidence for a model whereby AvrXa7 enters the host nucleus and mediates some aspect of host transcription. We will determine the capacity of AvrXa7 to bind DNA using standard protein expression techniques and DNA labeling methods. If specific binding motifs (consensus sequences) are determined, the ability of AvrXa7 to promote AvrXa7-dependent gene expression in yeast and plants will be assessed. AvrXa7 will be engineered to be expressed in rice plants using particle bombardment methodologies, and genes whose expression is affected by the presence of AvrXa7 in the rice plant will be identified. Insight into the strategies of pathogens may be gained if the targets of the avirulence proteins, acting in their virulence capacity, can be identified. Knowledge of the critical features of virulence determinants and their targets will undoubtedly lead to new genetically deployed strategies for crop protection. From a broader perspective, X. oryzae pv. oryzae is the most economically and most important bacterial pathogen of rice.

NON-TECHNICAL SUMMARY: The product of the avrXa7 gene (AvrXa7) is secreted by the bacterium Xanthomonas oryzae pv. oryzae into the cells of the host plant rice during infection and is required for pathogenicity.

PROGRESS: 2002/01 TO 2002/12
Bacterial avirulence genes are secreted by a Type III secretion system and control the elicitation of resistance in plants if the plant contains the corresponding gene for resistance. The gene avrXa7 is a member of the avrBs3 avirulence gene family, which contains features in the encoded protein that have the characteristics of plant transcription factors including an acidic transcriptional activation domain. AvrXa7 also controls the degree of virulence of the bacterial strain. We have identified related genes from a range of strains that have a significant contribution to virulence in each strain. The evidence indicates that bacterial blight of rice is highly dependent on a specific type of effector protein. Microarray hybridization experiments have provided candidate genes that may be elevated in transcription in the presence of AvrXa7. In addition, related genes were identified in whole genome analyses of related pathogen Xanthomons axonopodis pv. citri.

IMPACT: 2002/01 TO 2002/12
The studies afforded a better understanding of the mechanisms of plant resistance to disease and provided insight into new strategies for control of diseases.

PUBLICATIONS: 2002/01 TO 2002/12
1. Yang, B. L. B. Johnson, and F.F.White. 2002. Avirulence and virulence effectors from X. oryzae pv. oryzae. International Rice Research Conference, Sept. 16-20., Beijing, CN.
2. da Silva,A.C.R., Ferro,J.A., Reinach,F.C. et al. 2002. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417: 459-463.

PROJECT CONTACT:

Name: White, F. F.
Phone: 785-532-1362
Fax: 785-5325692
Email: fwhite@plantpath.ksu.edu

Item No. 7 of 12

ACCESSION NO: 0186416 SUBFILE: CRIS
PROJ NO: KS9663 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 2001-35319-09902 PROPOSAL NO: 2000-02849
START: 01 NOV 2000 TERM: 31 OCT 2002 FY: 2001 GRANT YR: 2001
GRANT AMT: $90,000

INVESTIGATOR: Warren, R. F.; Zhou, J.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

ROLES OF PATHOGEN-INDUCIBLE EREBP-LIKE SEQUENCES IN DISEASES RESISTANCE

OBJECTIVES: 1. Genetic characterization and mapping of nho mutants (nho1409 and nho1531, newly named nho1 and nho2, respectively). We will confirm that whether nho1 and nho2 are two distinct loci and determine the chromosomal locations of the two genes. 2. Examining interactions of nonhost resistance with other defense pathways. We will focus on nho1 and nho2 for their responses to different bacterial pathogens. The analysis will establish whether the general resistance conferred by the two NHO genes is targets of type III effector proteins. An answer to this question is of great importance to our understanding of plant-microbe interaction in general and bacterial pathogenesis in particular.

APPROACH: To accomplish our first goal, we used DNA macroarrays to identify genes that showed altered expression six hours after infection with Pseudomonas syringae strain DC3000 carrying the avirulence gene avrB. The plants used are genetically resistant to this pathogen strain. Potential EREBP-like sequences were identified among the Arabidopsis Expressed Sequence Tag (EST) collection by conducting BLAST searches using the conserved AP2 domain from the tomato gene Pti5 as a query sequence. DNA from these ESTs was blotted in duplicate and probed with PolyA mRNA isolated from plants that were vacuum-infiltrated with 2 x 10 cfu/mL of Pseudomonas or 10mM MgCl. Differential expression was present for 10 independent EST sequences. We were able to detect and confirm that six genes exhibited altered expression compared to mock-inoculated controls by Northern blot analysis. We are currently testing other pathogen strains to determine if this response is common or pathogen strain dependent. Additionally,we are testing several compounds that have been implicated as signals for the induction of pathogen resistance in plants to determine their effect on these identified EREBPs. These compounds include salicylic acid, jasmonic acid, and ethylene. Plants will be exposed or vacuum-infiltrated with these compounds & expression of EREBPs will be monitored over time by Northern hybridization. We will also be monitoring RNA expression in order to accomplish our second goal. However, these experiments will utilize a variety of Arabidopsis mutants. These mutants include those that exhibit increased suscept- ibility to pathogens and those that exhibit enhanced resistance to pathogens. Additionally, accumulation of EREBP mRNAs will be examined in plants that are non-responsive to the compounds mentioned previously. The gene products represented by the different mutants are positioned in signal transduction pathways that lead to pathogen resistance. Our final goal will utilize the Arabidopsis Knockout Facility and multiple rounds of PCR screening of DNA pools in order to identify at least 1 EREBP that contains a T-DNA insert. The validity of an identified T-DNA line will be confirmed by probing DNA blots, sequencing, and, assuming a knockout provides a characterizable phenotype, transforming a wild-type gene into mutant plants. The nature of the mutation will be determined in back crossed plants. Pathogen resistance in mutant plants will be assessed by visible symptoms, quantitative bacterial growth curves, and an HR assay. Finally, we may attempt to overexpress specific EREBPs in plants and determine if pathogen resistance is affected.

NON-TECHNICAL SUMMARY: We will be using molecular genetic approaches to define the roles of ethylene-responsive element binding proteins (EREBPs) in conferring disease resistance in plants.

PROGRESS: 2000/11 TO 2002/10
Ethylene-responsive element binding factors (ERFs or EREBPs) are transcription factors that are unique to plants and are involved in a plant's response to biotic and abiotic stress. Clearly, different ERF genes have diverse roles in mediating pathogen defense signal transduction pathways. ERFs may regulate different defense responses or may differ in the extent or timing of defense-gene induction. Detailed knowledge of ERF signal transduction pathways will require identification of ERFs involved in pathogen defense. To that end, we have characterized five Arabidopsis ERFs that exhibited enhanced transcript induction in response to pathogen infection.

IMPACT: 2000/11 TO 2002/10
The work systematically studied the regulation of Arabidopsis ERF genes in response to pathogen. Several candidate ERF genes have been identified that may play a role in plant disease resistance. The isolation of these genes may enhance our ability to improve disease resistance in crop plants.

PUBLICATIONS: 2000/11 TO 2002/10
Jianxiong Li, Libo Shan, Jian-Min Zhou, and Xiaoyan Tang (2002). Overexpression of Pto induces a salicylate-independent cell death but inhibits necrotic lesions caused by salicylate deficiency in tomato plants. MPMI, 15, 654-661.

PROJECT CONTACT:

Name: Warren, R. F.
Phone: 785-532-2415
Fax: 785-532-5692
Email: rwarren@plantpath.ksu.edu

Item No. 8 of 12

ACCESSION NO: 0189522 SUBFILE: CRIS
PROJ NO: KS9689 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2001-35319-10895 PROPOSAL NO: 2001-02621
START: 15 SEP 2001 TERM: 14 SEP 2004 GRANT YR: 2001
GRANT AMT: $300,000

INVESTIGATOR: Zhou, J. M.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

GENERAL RESISITANCE MEDIATED BY ARABIDOPSIS NHO GENES AND PSEUDOMONAS VIRULENCE

OBJECTIVES: 1. Isolation of NHO1 and NHO2. 2. Characterization of genetic interactions of NHO-mediated resisitance with bacterial virulence. 3. Molecular characterization of NHO1 and NHO2 genes.

APPROACH: The proposed research will elucidate plant mechanisms for innate immunity to nonhost pathogens, of which little is known. This in turn will lead to a better understanding of the molecular basis for host specificity in plant pathogen interactions and virulence mechanisms of bacterial pathogens. Furthermore, the isolation of NHO genes will advance the agricultural practices by exploiting the most abundant genetic resources of disease resisitance that still remain untapped.

NON-TECHNICAL SUMMARY: The long-term goal of this research is to understand the mechanisms of plant nonhost resisitance in the context of the host-pathogen interaction specificity.

PROGRESS: 2002/01 TO 2002/12
One aspect of Zhou's research focuses on nonhost disease resistance mechanisms in plants. It is poorly understood why a particular plant species is resistant to the vast majority of potential pathogens that infect other plant species, a phenomenon referred to as nonhost resistance. Here we show that Arabidopsis NHO1, encoding a glycerol kinase, is required for resistance to and induced by Pseudomonas syringae isolates from bean and tobacco. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance. Strikingly, P. s. pv. tomato DC3000, an isolate fully virulent on Arabidopsis, actively suppressed the NHO1 expression. This suppression is abolished in coi1 plants indicating that DC3000 required an intact jasmonic acid signaling pathway in the plant to suppress NHO1 expression. Constitutive overexpression of NHO1 led to enhanced resistance to this otherwise virulent bacterium. The presence of avrB in DC3000, which activates a cultivar-specific gene-for-gene resistance in Arabidopsis, restored the induction of NHO1 expression. Thus NHO1 is deployed for both general and specific resistance in Arabidopsis and targeted by the bacterium for parasitism.

IMPACT: 2002/01 TO 2002/12
This is the first nonhost resistance gene to be isolated from plants. The work establishes KSU as a leader in nonhost disease resistance studies. The continued research efforts will enable the much needed durable resistance to be used in major crop plants.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

PROJECT CONTACT:

Name: Zhou, J. M.
Phone: 785-532-2415
Fax: 785-532-5692
Email: Jzhou@plantpath.ksu.edu

Item No. 9 of 12

ACCESSION NO: 0193269 SUBFILE: CRIS
PROJ NO: KS9719 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2002-35319-12517 PROPOSAL NO: 2002-02349
START: 01 SEP 2002 TERM: 31 AUG 2005 GRANT YR: 2002
GRANT AMT: $271,000

INVESTIGATOR: White, F.; Johnson, L.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

GENOMIC ANALYSIS AND CLONING OF THE DISEASE RESISTANCE GENE XA7

OBJECTIVES: The specific objectives are: (1) Isolation and mapping of sequence tagged markers that are linked to Xa7; (2) molecular identification and cloning of Xa7; and, (3) characterization of the interaction of Xa7 with the AvrXa7 protein.

APPROACH: Short sequence regions of the rice genome in the vicinity of Xa7 will be amplified from the parental lines IRBB7 (Xa7) and IR24. The sequences will be chosen from the available sequence for chromosome 6 of rice genotypes that do not have Xa7. The amplified sequences will be analyzed by gel electrophoresis and DNA sequencing for polymorphisms between the parental lines. Polymorphic sites will be mapped relative to Xa7 and each other. Sites that map within close proximity to Xa7will be used as probes for retrieval of cosmid clones. Additional markers will be developed from the sequence of the cosmid DNA. Candidate genes from either the cosmid DNA or related DNA from the databases will be tested in transient assays for Xa7 activity. Candidate clones will be stably transferred to susceptible rice line and progeny will be tested for Xa7 activity. The nature of the interaction of either the gene for Xa7 or the gene product with the elicitor AvrXa7 will be explored by protein binding and gene transcription assays.

NON-TECHNICAL SUMMARY: Bacterial blight of rice is a major bacterial disease of rice and one of the most importance bacterial diseases of agronomic crop production. Resistance to bacterial infection in rice lines harboring the resistance gene Xa7 is elicited by bacteria of the species Xanthomonas oryzae pv. oryzae that harbor the gene avrXa7.

PROGRESS: 2002/01 TO 2002/12
Bacterial blight of rice is a major disease of rice and one of the most important bacterial diseases of agronomic crop production. Resistance to bacterial infection in culitvars harboring the resistance gene Xa7 is elicited by the species Xanthomonas oryzae pv. oryzae harboring the gene avrXa7. The gene avrXa7 directs the synthesis of the protein AvrXa7. AvrXa7 must be secreted by the bacteria and enter the plant cells where evidence indicates the protein must further travel to the nucleus of the host cell in order to elicit the resistance reaction. In this regard, Xa7 appears to mediate resistance in a manner different from other resistance genes. AvrXa7 is also an important factor for the virulence of bacteria that harbor the gene for the protein. Thus, Xa7 is targeted to a virulence factor of the pathogen. The pathogen population must produce a variant of an important virulence factor, if the population is to avoid eliciting resistance in plants with Xa7 and continue to thrive in rice growing regions. Cloning and characterization of Xa7 will be required for further understanding of the mechanism, which, in turn, may permit improved strategies for resistance gene deployment. We propose to clone Xa7 by identifying markers that are linked to the gene. The markers will consist of DNA sequences in the genome near Xa7 that differ between susceptible and resistant parents. Understanding the mechanism of the Xa7/AvrXa7 interaction will assist in strategies to improve the resistance of rice and other crop plants to disease.

IMPACT: 2002/01 TO 2002/12
The studies afforded a better understanding of the mechanisms of plant resistance to disease and provided insight into new strategies for control of diseases.

PUBLICATIONS: 2002/01 TO 2002/12
Porter, BW, JM Chittoor, Myano, T Sasaki, and FF. White. 2002. Development and mapping of markers to the rice bacterial leaf blight resistance gene Xa7. Crop Science. In press.

PROJECT CONTACT:

Name: White, F.
Phone: 785-5321362
Fax: 785-5325692
Email: fwhite@ksu.edu

Item No. 10 of 12

ACCESSION NO: 0186039 SUBFILE: CRIS
PROJ NO: KS9763 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT
CONTRACT/GRANT/AGREEMENT NO: 2001-35302-09972 PROPOSAL NO: 2000-02963
START: 15 NOV 2000 TERM: 14 NOV 2002 FY: 2001 GRANT YR: 2001
GRANT AMT: $116,000

INVESTIGATOR: Clem, R. J.

PERFORMING INSTITUTION:
BIOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

APOPTOSIS AS AN ANTI-VIRAL DEFENSE MECHANISM IN INSECTS

OBJECTIVES: Directly determine whether apoptosis is responsible for decreased infectivity of Autographa californica nucleopolyhedrovirus mutants lacking the p35 gene in Spodoptera frugiperda larvae. Characterize the effects of apoptosis on baculovirus replication and pathogenesis in S. frugiperda. Compare the ability of p35 and other viral anti-apoptotic genes, such as iap genes, to support virus replication in vivo.

APPROACH: Larvae from two species, Spodoptera frugiperda and Trichoplusia ni, will be infected with wild type and p35 mutant viruses expressing the marker gene eGFP. Larvae will be infected by either oral inoculation or intrahemocelic injection, and the presence of apoptotic cells in specific tissues of interest will be determined by TUNEL assay. The progress of viral infection will be simultaneously tracked using the eGFP marker. Levels of virus replication will be monitored and the level of apoptosis will be correlated with the level of viral replication and lethality. Recombinant viruses expressing other viral anti-apoptotic genes will also be used in similar assays.

NON-TECHNICAL SUMMARY: Pest insect populations are naturally limited by a number of factors. One such factor is the presence of pathogenic agents such as bacteria, fungi, nematodes and viruses. Although all insects are susceptible to infection by one or more pathogenic agents, they are able to resist most agents quite effectively due to a highly effective innate immune system. There is considerable interest in utilizing insect pathogens as biological control agents to safely curtail insect populations while avoiding the harmful ecological effects of chemical pesticides. However, little is known about the molecular mechanisms utilized by the insect immune system to defeat viruses. An improved fundamental understanding of the insect immune system and how it interacts with viral pathogens will aid in our ability to control pest insects through the development of improved viral biological control agents.

PROGRESS: 2000/11 TO 2002/11
This basic research project was designed to yield information concerning the role of apoptosis, or cellular suicide, in the immune system of insects. The insect immune system is highly effective against a variety of potential pathogens that insects encounter continuously, such as viruses, bacteria, and fungi. While there is a fairly good basic understanding of how the insect immune system functions against bacteria and fungi, currently little is known about how the insect immune system works against viruses. We have shown in the past that one type of insect viruses, called baculoviruses, induce insect cells to undergo apoptosis in culture. The goal of this project was to further characterize the effects of an apoptotic response on the ability of baculovirus to infect and replicate in lepidopteran insects. Our results demonstrate that apoptosis occurs in the tissues of the fall armyworm (Spodoptera frugiperda) infected with a mutant of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) that induces apoptosis in cultured cells. Apoptosis severely limits the ability of AcMNPV to establish infection in S. frugiperda larvae, as well as the ability of the infection to spread within the insect. In addition, we found that hemocytes are not significantly involved in the pathogenesis of AcMNPV in S. frugiperda, a somewhat surprising result. Finally, our studies showed that expression of a DNA repair enzyme can improve the ability of AcMNPV to resist the effects of UV light, and thus is a potential strategy to reduce UV inactivation in a viral pesticide.

IMPACT: 2000/11 TO 2002/11
Pest insects are responsible for huge economic losses, necessitating the widespread use of chemical pesticides. Thus more environmentally friendly methods for controlling pest insects are needed. Biological control agents such as baculoviruses represent a promising method for helping to control pest insect populations while reducing the need for chemical pesticides. In order to develop more effective biological control agents, a better basic understanding of the insect immune system is needed. The results of these studies shed light on how the insect immune system fights off viral pathogens, and may be useful in developing technologies for controlling pest insects that are more effective and safe than those currently used.

PUBLICATIONS: 2000/11 TO 2002/11
Clarke, T.E. and R.J. Clem. 2002. Lack of involvement of hemocytes in the establishment and spread of infection in Spodoptera frugiperda larvae infected with the baculovirus AcMNPV by intrahemocoelic injection. J. Gen. Virol. 83:1565-1572.

PROJECT CONTACT:

Name: Clem, R. J.
Phone: 785-532-3172
Fax: 785-532-6653
Email: rclem@ksu.edu

Item No. 11 of 12

ACCESSION NO: 0189236 SUBFILE: CRIS
PROJ NO: KS9783 AGENCY: CSREES KAN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2002-35319-11655 PROPOSAL NO: 2001-02618
START: 01 DEC 2001 TERM: 30 NOV 2003 GRANT YR: 2002
GRANT AMT: $131,000

INVESTIGATOR: Shah, J.

PERFORMING INSTITUTION:
BIOLOGY
KANSAS STATE UNIV
MANHATTAN, KANSAS 66506

LIPID SIGNALING AND NPR1-INDEPENDENT PATHWAY IN PLANT DEFENSE

OBJECTIVES: The Arabidopsis thaliana SSI2 protein is an important regulator/modulator of NPR1-independent plant defense responses. A missense mutation (ssi2-1) in the SSI2 encoded stearoyl-ACP desaturase activity results in altered expression of the pathogenesis-related (PR) and PDF1.2 defense genes, and resistance against various pathogens. The ssi2-1 mutant therefore provides an important tool for further dissection of the role of lipids in plant defense. We propose to use the power of genetics to further evaluate the role of lipid signaling in plant defense. Four major objectives will be pursued: 1. Epistatic interaction between ssi2-1 and pad4-1 and eds5-1 2. Genetic characterization of supssi2 mutants 3. Resistance of supssi2 mutants to pathogens 4. Map-based cloning of supssi2 genes.

APPROACH: 1.Epistatic interaction between ssi2-1 and pad4-1 and eds5-1: We will characterize the epistatic interaction between the ssi2-1 mutant and the pad4-1 and eds5-1 mutants. Mutations in the eds5-1 and pad4-1 plants compromises defense gene expression and resistance to various pathogens. Double and triple mutants will be generated and evaluated by RNA gel blot analysis for defense gene expression and for resistance to bacterial and fungal pathogens. 2.Genetic characterization of supssi2 mutants: We have identified three, second-site suppressors of ssi2-1 (supssi2). Some of these supssi2 mutants might define other components of lipid signaling pathways in plant defense. Genetic analysis will be carried out to determine the dominant/recessive nature of the mutant alleles. The mutants will be crossed to each other and placed into complementation groups. We will characterize expression of defense genes in response to various biotic and abiotic stresses in these supssi2 mutants. 3.Resistance of supssi2 mutants to pathogens The supssi2 mutants, and as control the ssi2-1 mutant and wild type plants, will be infected with various bacterial and fungal pathogens to determine the susceptibility/resistance of the supssi2 mutants to these pathogens 4. Map-based cloning of supssi2 genes: The supssi2 mutants will be fine-mapped and the genes cloned and sequenced. The cloned genes will be transformed into plants to confirm the identity of the gene. The expression of these supssi2 genes will be studied in response to various biotic and abiotic stresses. Identifying the components and understanding the mechanism of signaling through this uncharacterized SSI2-regulated defense pathway will assist in devising alternative strategies of plant protection.

NON-TECHNICAL SUMMARY: Diseases severely limit plant productivity and quality. Fungicides and pesticides target a broad-spectrum of pathogens and pests. However, this kind of chemical control is expensive and harmful to the environment and public health. Plants have preformed and inducible defense mechanisms

PROGRESS: 2002/01 TO 2002/12
Lipids and lipid-derived molecules play important signaling roles in plant defense responses. A complex network of cross talk between signaling pathways involving the plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene regulate plant defense responses. Genetic and molecular approaches have been taken to understand the mechanism of SA action in plant defense against disease. The Arabidopsis thaliana NPR1 gene is one of the key components of the SA signal transduction pathway in plant defense. However, our recent studies of the ssi1, ssi2 and ssi4 mutants suggest the presence of an alternative NPR1-independent pathway for mediating SA responses (Shah et al., 2001; Yoshioka et al., 2001; Shirano et al., 2002). A patent application on this novel pathway has been filed jointly by Kansas State University and Boyce Thompson Institute. The SSI2 gene encodes an enzyme that plays important role in lipid signaling in plants (Kachroo et al., 2001). We also have evidence implicating the SSI1 and SSI2 gene in mediating cross talk between the SA and JA pathways. In order to identify how SSI2 and lipids regulate cross talk between the different defense signaling pathways we have identified seven suppressors (sfd) of the ssi2 mutant, which abolish the ssi2-conferred phenotypes. These seven sfd mutants define four complementation groups. Mutants in all four complementation groups suppress ssi2-conferred cell death, dwarfing, NPR1-independent PR-1 expression and resistance to the biotrophic pathogen, Pseudomonas syringae pv maculicola. In addition, the sfd1 and sfd3 mutants also reverse the ssi2-conferred block on JA-activated expression of the defensin gene. However, none of them suppress ssi2-conferred susceptibility to the necrotrophic pathogen Botrytis cinerea. The common, alteration in all sfd mutants is the lowering of 16:3 fatty acid levels, suggesting that polyunsaturated fatty acid derived molecules and signaling may play important role in plant defense responses. The SFD1 gene has been cloned and is predicted to encode a glycerol-3-phosphate dehydrogenase implicating the role of glycerol-3-phosphate fluxes in regulating chloroplast lipid biosynthesis. These studies should now allow us to further address the involvement of these fatty acids in plant defense response.

IMPACT: 2002/01 TO 2002/12
In addition to their contribution to cell integrity as an important constituent of cell membranes, lipids and lipid-derived molecules are important signaling molecules in plant development and response to stress. Understanding lipid dynamics, biosynthesis and signaling is important for improving our understanding of plant growth, development and response to environment. In addition, some steps in lipid metabolism may be important targets for developing plants with improved stress tolerance.

PUBLICATIONS: 2002/01 TO 2002/12
Takahashi, H., Miller, J., Nozaki, Y., Sukamoto, Takeda, M., Shah, J., Hase, S., Ikegami, M., Ehara, Y., and Dinesh-Kumar, S.P. (2002) RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene, conferring resistance to cucumber mosaic virus requires salicylic acid, ethylene and a novel signal transduction mechanism. The Plant J. 32, 655-667.

PROJECT CONTACT:

Name: Shah, J.
Phone: 785-532-6360
Fax: 785-532-6653
Email: shah@ksu.edu

Item No. 12 of 12

ACCESSION NO: 0181195 SUBFILE: CRIS
PROJ NO: KANR-9802493 AGENCY: CSREES KANR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 98-35302-6820
START: 15 DEC 1998 TERM: 31 DEC 2001 FY: 2001 GRANT YR: 1999
GRANT AMT: $190,000

INVESTIGATOR: Kramer, K. J.

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
MANHATTAN, KANSAS 66506

IMPROVEMENT OF INSECT CHITINASE AS A BIOPESTICIDE IN TRANSGENIC PLANTS

OBJECTIVES: 9802493. The objectives are to extend the insect chitinase-tobacco plant transformation work completed under the previous NRI award to other plants including wheat, rice and sorghum, and also to conduct structure-function studies of the insect chitinase protein so that improved and more effective chitinase-based biopesticides can be genetically engineered in plants.

APPROACH: The study will generate transgenic cereals containing the insect chitinase transgene. A synthetic version of the chitinase gene with plant-preferred codons will be introduced into wheat, rice, and sorghum using Agrobacterium-mediated transformation. Transformed plants expressing high levels of the chitinase protein will be identified by Southern and western blot analyses. Homozygous progeny obtained from these plants will be used for bioassays. Transgenic plants will be tested for resistance to feeding damage by insect pests of that particular crop, including the yellow stem borer and fall armyworm for rice, fall armyworm for wheat, green bug and fall armyworm for sorghum, and stored grain insects for the seeds. This study also will characterize deletion and point mutants of insect chitinase. Three deletion mutants and several point mutants are already generated and additional ones will be used for the expression and purification of mutant forms of the enzyme.

PROGRESS: 2000/10 TO 2001/09
Aim 1. Characterization of point mutants of insect chitinases. In an attempt to generate chitinases with improved insecticidal properties, deletion mutagenesis was carried out to generate altered forms of insect chitinase. Insect chitinase is divided into an N-terminal signal peptide, a middle catalytic domain, a serine-threonine-rich domain and a C-terminal cysteine-rich domain. To understand the specific functions of these domains, we have created several deletions of the insect chitinase gene involving one or more of these domains so that function of each domain could be demonstrated. In addition, we have constructed additional modified genes in which a chitin binding domain from a plant (rice) or an extra copy of the cysteine-rich domain from insect chitinase have been added. Some of these deletion/addition mutant genes have been utilized to express the corresponding proteins in the insect cell/baculovirus expression system. The purified enzymes have been assayed for enzyme activity, substrate specificity and chitin-binding ability. These studies have reveled that the catalytic activity remain in the N-terminal 400 amino acid segment and that the C-terminal cysteine-rich region is involved in chitin-binding. Studies with the other deletion/addition constructs are in progress. Aim 2. Generation of transgenic cereal plants with insect chitinase gene. The insect chitinase gene has been introduced into several crop plants including wheat, soybean and corn. The presence of the transgene has been confirmed by PCR and by Southern blotting experiments. Expression of the gene has been confirmed by Northern blot anlysis in a few lines. Western blot analysis has yielded mixed results. There is low level expression in some wheat and corn plants and we are trying to identify some lines with high level expression. More recently in cooperation with Troy Weeks of USDA-ARS in Lincoln, NE, we have analyzed several putative transgenic sorghum plants for the expression of insect chitinase. One line appears to have a high level expression. Molecular analysis of this plant is in progress. Plans for the coming year: The expression constructs with different domains of the insect chitinase gene will be utilized to obtain large amounts of the corresponding proteins. These proteins will be used in enzyme assays, in vitro antifungal assays and in vivo assays for insect growth inhibition to determine the efficacy of each preparation. The transgenic plants with high level expression of insect chitinase gene will be propagated to obtain progeny plants which will then be tested for resistance to appropriate target insects.

IMPACT: 2000/10 TO 2001/09
The elucidation of the role of different residues in the conserved region II of insect chitinase and the functions of specific domains of this enzyme in catalysis, chitin binding and stability has yielded valuable concerning the type of gene construct that we need to utilize in transformation of crop plants for enhanced resistance to insects.

PUBLICATIONS: 2000/10 TO 2001/09
1. Zhu, X., Zhang, H., Fukamizo, T.,Muthukrishnan, S., and Kramer, K.J. 2001. Properties of Manduca sexta Chitinase and its C-Terminal Deletions. Insect Biochem. Molec. Biol. 31:1221-1230.
2. Muthukrishnan, S., Kramer, K. J., Zhang, H., Lu, Y., and Matsumiya, M. and Anand, A. (2001) Use of Chitinases as Biopesticides. In: Chitin and Chitosan - Chitin and Chitosan in Life Science (ed. T. Uragami, K. Kurita & T. Fukamizo). Kodansha Scientific Ltd. ISBN 4-906464-13-0. pp. 415-418.